Attenuated mutant dengue viruses comprising a mutation in the NS4B non-structural protein

ABSTRACT

The invention features novel attenuated dengue virus mutants and compositions thereof. The invention further features an attenuated dengue virus comprising a proline to leucine change at amino acid position 2343 of non-structural protein 4B (NS4B), wherein the numbering is based upon the prototypic isolate DEN4 Dominica 1981 and the attenuated mutant DEN virus has at least one of the following properties: improved replication in Vero cells or restricted replication in mosquito cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/240,849, filed Sep. 22, 2011, now granted as U.S. Pat. No. 8,632,782,which is a divisional of U.S. application Ser. No. 12/396,376, filedOct. 18, 2011, now granted as U.S. Pat. No. 8,039,003, which is acontinuation of U.S. application Ser. No. 11/446,050, filed Jun. 2,2006, now granted as U.S. Pat. No. 7,560,118, which is a division ofSer. No. 10/719,547, filed Nov. 21, 2003, now granted as U.S. Pat. No.7,226,602, which is continuation and claims the benefit of priority ofInternational Application No. PCT/US2002/16308, filed May 22, 2002,designating the United States of America and published in English as WO2002/095075 on Nov. 28, 2002, which claims the benefit of priority ofU.S. Provisional Application No. 60/293,049, filed May 22, 2001, thedisclosure of each of which is incorporated herein by reference in itsentirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 12, 2014, isnamed 84405DIV3_ST25.txt and is 274,432 bytes in size.

FIELD OF THE INVENTION

A menu of mutations was developed that is useful in fine-tuning theattenuation and growth characteristics of dengue virus vaccines.

BACKGROUND OF THE INVENTION

Dengue virus is a positive-sense RNA virus belonging to the Flavivirusgenus of the family Flaviviridae. Dengue virus is widely distributedthroughout the tropical and semitropical regions of the world and istransmitted to humans by mosquito vectors. Dengue virus is a leadingcause of hospitalization and death in children in at least eighttropical Asian countries (WHO, 1997. Dengue haemorrhagic fever:diagnosis, treatment prevention and control—2nd ed. Geneva: WHO). Thereare four serotypes of dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4)which annually cause an estimated 50-100 million cases of dengue feverand 500,000 cases of the more severe form of dengue virus infection,dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (Gubler, D. J.& Meltzer, M. 1999 Adv Virus Res 53:35-70). DHF/DSS is seenpredominately in children and adults experiencing a second dengue virusinfection with a serotype different than that of their first denguevirus infection and in primary infection of infants who still havecirculating dengue-specific maternal antibody (Burke, D. S. et al. 1988Am J Trop Med Hyg 38:172-80; Halstead, S. B. et al. 1969 Am J Trop MedHyg 18:997-1021; Thein, S. et al. 1997 Am J Trop Med Hyg 56:566-72). Avaccine is needed to lessen the disease burden caused by dengue virus,but none is licensed. Because of the association of more severe diseasewith secondary dengue virus infection, a successful vaccine must induceimmunity to all four serotypes. Immunity is primarily mediated byneutralizing antibody directed against the envelope E glycoprotein, avirion structural protein. Infection with one serotype induceslong-lived homotypic immunity and a short-lived heterotypic immunity(Sabin, A. 1955 Amer J Trop Med Hyg 4:198-207). Therefore, the goal ofimmunization is to induce a long-lived neutralizing antibody responseagainst DEN-1, DEN-2, DEN-3, and DEN-4, which can best be achievedeconomically using live attenuated virus vaccines. This is a reasonablegoal since a live attenuated vaccine has already been developed for therelated yellow fever virus, another mosquito-borne flavivirus present intropical and semitropical regions of the world (Monath, T. P. & Heinz,F. X. 1996 in: Fields B. N. et al. eds. Fields Virology Philadelphia:Lippincott-Ravan Publishers, 961-1034).

Several live attenuated dengue vaccine candidates have been developedand evaluated in humans or non-human primates. The first live attenuateddengue vaccine candidates were host range mutants developed by serialpassage of wild type dengue viruses in the brains of mice and selectionof mutants attenuated for humans (Kimura, R. & Hotta, S. 1944 Japanese JBacteriology 1:96-99; Sabin, A. B. & Schlesinger, R. W. 1945 Science101:640; Wisseman, C. L. Jr. et al. 1963 Am J Trop Med 12:620-623).Although these candidate vaccine viruses were immunogenic in humans,their poor growth in cell culture discouraged further development.Additional live attenuated DEN-1, DEN-2, DEN-3, and DEN-4 vaccinecandidates have been developed by serial passage in tissue culture(Angsubhakorn, S. et al. 1994 Southeast Asian J Trop Med Public Health25:554-9; Bancroft, W. H. et al. 1981 Infect Immun 31:698-703;Bhamarapravati, N. et al. 1987 Bull World Health Organ 65:189-95;Eckels, K. H. et al. 1984 Am J Trop Med Hyg 33:684-9; Hoke, C. H. Jr. etal. 1990 Am J Trop Med Hyg 43:219-26; Kanesa-thasan, N. et al. 2001Vaccine 19:3179-88) or by chemical mutagenesis (McKee, K. T. Jr. et al.1987 Am J Trop Med Hyg 36:435-42). It has proven very difficult toachieve a satisfactory balance between attenuation and immunogenicityfor each of the four serotypes of dengue virus using these approachesand to formulate a tetravalent vaccine that is safe and satisfactorilyimmunogenic against each of the four dengue viruses (Kanesa-thasan, N.et al. 2001 Vaccine 19:3179-88; Bhamarapravati, N. & Sutee, Y. 2000Vaccine 18 Suppl 2: 44-7).

Two major advances utilizing recombinant DNA technology have recentlymade it possible to develop additional promising live attenuated denguevirus vaccine candidates. First, methods have been developed to recoverinfectious dengue virus from cells transfected with RNA transcriptsderived from a full-length cDNA clone of the dengue virus genome, thusmaking it possible to derive infectious viruses bearing attenuatingmutations which have been introduced into the cDNA clone bysite-directed mutagenesis (Lai, C. J. et al. 1991 PNAS USA 88:5139-43).Second, it is possible to produce antigenic chimeric viruses in whichthe structural protein coding region of the full-length cDNA clone ofdengue virus is replaced by that of a different dengue virus serotype orfrom a more divergent flavivirus (Bray, M. & Lai, C. J. 1991 PNAS USA88: 10342-6; Chen, W. et al. 1995 J Virol 69:5186-90; Huang, C. Y. etal. 2000 J Virol 74:3020-8; Pletnev, A. G. & Men, R. 1998 PNAS USA95:1746-51). These techniques have been used to construct intertypicchimeric dengue viruses which have been shown to be effective inprotecting monkeys against homologous dengue virus challenge (Bray, M.et al. 1996 J Virol 70:4162-6). Despite these advances, there is a needto develop attenuated antigenic dengue virus vaccines that specify asatisfactory balance between attenuation and immunogenicity for humans.

SUMMARY OF THE INVENTION

The invention provides mutations that confer temperature sensitivity inVero cells or human liver cells, host-cell restriction in mosquito orhuman liver cells, host-cell adaptation for improved replication in Verocells, or attenuation in mice, which mutations are useful in fine tuningthe attenuation and growth characteristics of dengue virus vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows growth of wt DEN4 2A and vaccine candidate, 2AΔ30, in Veroand HuH-7 cells. Vero (A) or HuH-7 (B) cells were infected with DEN4 2Aor 2AΔ30 at a multiplicity of infection (MOI) of 10 or 0.01. Confluentcell monolayers in 25-mm tissue culture flasks were washed and overlaidwith a 1.5 ml inoculum containing the indicated virus. After a two hourincubation at 37° C., cells were washed three times in PBS and 7 ml ofculture media supplemented with 2% FBS was added. A 1 ml aliquot oftissue culture medium was removed, replaced with fresh medium, anddesignated the 0 hour time-point. At the indicated time pointspost-infection, samples of tissue culture media were removed and frozenat −70° C. The level of viral replication was assayed by plaquetitration in Vero cells. Briefly, serial ten-fold dilutions of cellculture media samples were inoculated onto confluent Vero cellmonolayers in 24-well plates in duplicate and overlaid with Opti-MEMcontaining 0.8% methylcellulose. After five days, plaques werevisualized by immunoperoxidase staining as described in Example 1.

FIG. 2 shows generation of temperature-sensitive (ts) DEN4 viruses by5-fluorouracil (5-FU) chemical mutagenesis. The wild-type DEN4 2A viruswas derived from a cDNA clone of DEN4 strain 814669 (Dominica, 1981).Vero cells were infected with DEN4 2A and overlaid with culture mediacontaining 1 mM 5-fluorouracil (5-FU) which resulted in a reduction ofapproximately 100-fold in viral replication when compared to untreatedcontrols. Viral progeny from the 1 mM 5-FU-treated cultures weresubjected to a single round of terminal dilutions generating 1,248biologically cloned viruses which were screened for ts phenotypes byassessing virus replication at 35° C. and 39° C. in Vero and HuH-7cells. Virus clones which demonstrated a 100-fold or greater reductionin titer at 39° C. were terminally diluted an additional two times andamplified in Vero cells. Temperature-sensitive phenotypes of the 3×biologically cloned viruses were confirmed by evaluating efficiency ofplaque formation (EOP) in the indicated cells as described in Example 1.

FIG. 3 shows plaque size phenotypes of representative 5-FU mutant DEN4viruses. Serial ten-fold dilutions of wild-type DEN4 2A-13 (A), 5-FUmutant viruses #569 and #1189 (B), and 5-FU mutant viruses #1083 and#311 (C) were inoculated onto confluent Vero and HuH-7 cell monolayersin 24-well plates. After incubation at 35° C. for two hours, monolayerswere overlaid with 0.8% methylcellulose culture media. Followingincubation at 35° C. for five days, plaques were visualized byimmunoperoxidase staining. Viruses which had a plaque size that was ≦1mm (approximately ≦50% the size of wt DEN4 2A-13) at the permissivetemperature of 35° C. were designated as having the small-plaque (sp)phenotype. Mutant viruses #569 and #1189 (B) were sp in both Vero andHuH-7 cells, and #311 and #1083 (C) were sp in only HuH-7 cells.

FIG. 4 shows generation of recombinant DEN4 viruses. (A), The p4 cDNAclone is represented which was constructed from the 2A cDNA clone(derived from DEN4 814669) by site-directed mutagenesis. Restrictionenzyme sites were introduced or removed to facilitate subsequent cloningof DEN4 recombinants bearing introduced attenuating mutations.Restriction enzyme sites are shown and define fragments of the genomethat were sub-cloned into modified pUC-119 vectors for site-directedmutagenesis to introduce mutations identified in the 5-FU mutantviruses. (B), An outline of the methods used to generate rDEN4 virusesis also represented and described in Example 1.

FIG. 5 shows amino acid sequence of the rDEN4 NS5 gene (SEQ ID NO: 1).Eighty underlined amino acid pairs were mutagenized to alanine pairs; 32pairs in boldface represent mutant viruses that could be recovered ineither Vero or C6/36 cells; pairs in normal type represent mutantviruses that could not be recovered in either Vero or C6/36 cells. Boxedregions indicate putative functional domains, including anS-adenosylmethionine utilizing methyltransferase domain (SAM), animportin-β binding domain adjacent to a nuclear localization sequence(importin-β−binding+NLS) and an RNA-dependent RNA polymerase domain(Polymerase).

FIG. 6 shows plaque size of mutant 5-1A1 in C6/36 cells. Note that 5-1A1has a small plaque phenotype in C6/36 cells relative to that of the wildtype virus.

FIG. 7 shows growth of wild type rDEN4 and 5-1A1 in C6/36 cells. Cellswere inoculated in triplicate with each virus at an MOI of 0.01, and theamount of virus present in the supernatants that were harvested on theindicated days was determined by plaque enumeration in Vero cells. Thetiters are expressed as log₁₀ PFU/ml±standard error.

FIG. 8 shows nucleotide alignment of the 3′ UTR of mosquito-borne andtick-borne flaviviruses. cDNA sequences are shown 5′ to 3′ and representa portion of the UTR corresponding to DEN4 nucleotides 10417 to 10649(3′ genome end). Nucleotide numbering represents the position in thealignment. Regions deleted or swapped are indicated using the nucleotidenumbering of DEN4. GenBank accession numbers for mosquito-borne viruses:DEN4 (SEQ ID NO: 2): AF326825, DEN1 (SEQ ID NO: 3): U88535, DEN2 (SEQ IDNO: 4): AF038403, DEN3 (SEQ ID NO: 5): M93130, West Nile virus (WN) (SEQID NO: 6): M12294, Japanese encephalitis virus (JE) (SEQ ID NO: 7):AF315119, Yellow fever virus (YF) (SEQ ID NO: 8): U17067; GenBankaccession numbers for tick-borne viruses: Powassan virus (POW) (SEQ IDNO: 9): L06436, Louping Ill virus (LI) (SEQ ID NO: 10): Y07863,Tick-borne encephalitis virus (TBE) (SEQ ID NO: 11): U27495, and Langatvirus (LGT) (SEQ ID NO: 12): AF253419.

FIG. 9 shows genetic map of plasmid p4. Dengue cDNA is shown as boldline, with the C-prM-E region exchanged during construction of chimericdengue virus cDNAs indicated.

FIG. 10 shows plaque size phenotypes of rDEN4 viruses encoding Veroadaptation mutations. Serial three-fold dilutions of the indicatedviruses were inoculated onto confluent Vero and C6/36 cell monolayers in6-well plates. After incubation at 37° C. (Vero) or 32° C. (C6/36) fortwo hours, monolayers were overlaid with 0.8% methylcellulose culturemedia. Following incubation for five days, plaques were visualized byimmunoperoxidase staining. Values below each well are the average plaquesize in mm standard error. For each of the virus-infected wells, 36plaques were measured on the digital image of the 6-well plate on AdobePhotoshop at 300% view.

FIG. 11 shows growth curve in Vero cells of rDEN4 viruses encodingsingle Vero adaptation mutations. Vero cells were infected with theindicated viruses at an MOI of 0.01. Confluent cell monolayers in 25-cm²tissue culture flasks were washed and overlaid with a 1.5 ml inoculumcontaining the indicated virus. After a two hour incubation at 37° C.,cells were washed three times in PBS and 5 ml of culture mediumsupplemented with 2% FBS was added. A 1 ml aliquot of tissue culturemedium was removed, replaced with fresh medium, and designated the 0hour time-point. At the indicated time points post-infection, samples oftissue culture medium were removed, clarified, and frozen at −70° C. Thelevel of virus replication was assayed by plaque titration in Verocells. Briefly, serial ten-fold dilutions of cell culture media sampleswere inoculated onto confluent Vero cell monolayers in 24-well plates induplicate and overlaid with Opti-MEM containing 0.8% methylcellulose.After five days, plaques were visualized by immunoperoxidase staining asdescribed in Example 1. Limit of detection (L.O.D.) is ≧0.7 log₁₀PFU/ml.

FIG. 12 shows growth curve in Vero cells of rDEN4 viruses encodingcombined Vero cell adaptation mutations. Vero cells were infected withthe indicated viruses at an MOI of 0.01. Confluent cell monolayers in25-cm² tissue culture flasks were washed and overlaid with a 1.5 mlinoculum containing the indicated virus. After a two hour incubation at37° C., cells were washed three times in PBS and 5 ml of culture mediumsupplemented with 2% FBS was added. A 1 ml aliquot of tissue culturemedium was removed, replaced with fresh medium, and designated the 0hour time-point. At the indicated time points post-infection, samples oftissue culture medium were removed, clarified, and frozen at −70° C. Thelevel of virus replication was assayed by plaque titration in Verocells. Limit of detection (L.O.D.) is ≧0.7 log₁₀ PFU/ml.

BRIEF DESCRIPTION OF THE TABLES

Table 1.

Susceptibility of mice to intracerebral DEN4 infection is age-dependent.

Table 2.

Temperature-sensitive (ts) and mouse brain attenuation (att) phenotypesof 5-FU mutant DEN4 viruses.

Table 3.

Nucleotide and amino acid differences of the 5-FU mutant viruses whichare ts in both Vero and HuH-7 cells.

Table 4.

Nucleotide and amino acid differences of the 5-FU mutant viruses whichare ts in only HuH-7 cells.

Table 5.

Mutations which are represented in multiple 5-FU mutant DEN4 viruses.

Table 6.

Addition of ts mutation 4995 to rDEN4Δ30 confers a ts phenotype andfurther attenuates its replication in suckling mouse brain.

Table 7.

Temperature-sensitive (ts) and mouse brain attenuation (att) phenotypesof 5-FU DEN4 mutant viruses which exhibit a small plaque (sp) phenotype.

Table 8.

Viruses with both ts and sp phenotypes are more restricted inreplication in mouse brain than those with only a ts phenotype.

Table 9.

Nucleotide and amino acid differences of the 5-FU mutant DEN4 viruseswhich produce small plaques in both Vero and HuH-7 cells.

Table 10.

Nucleotide and amino acid differences of the 5-FU mutant DEN4 viruseswhich produce small plaques in only HuH-7 cells.

Table 11.

Putative Vero cell adaptation mutations derived from the full set of5-FU mutant viruses.

Table 12.

Mutagenic oligonucleotides used to generate recombinant DEN4 virusescontaining single 5-FU mutations.

Table 13.

sp, ts and mouse attenuation phenotypes of rDEN4 mutant viruses encodingsingle mutations identified in six sp 5-FU mutant viruses.

Table 14.

Phenotypes of rDEN4 mutant viruses encoding single mutations identifiedin 10 5-FU mutant viruses that are ts in both Vero and HuH-7 cells.

Table 15.

sp, ts and mouse attenuation phenotypes of rDEN4 mutant viruses encodingsingle mutations identified in 3 HuH-7 cell-specific ts 5-FU mutantviruses.

Table 16.

Temperature-sensitive (ts) and mouse brain attenuation (att) phenotypesof additional rDEN4 viruses encoding single 5-FU mutations.

Table 17.

Growth of wt DEN-4 2A-13 in SCID mice transplanted with HuH-7 cells.

Table 18.

Combination of ts mutations, NS3 4995 and NS5 7849, in rDEN4 results inan additive ts phenotype.

Table 19.

The 5-FU mutations are compatible with the Δ30 mutation for replicationin the brain of suckling mice.

Table 20.

Temperature-sensitive and mouse brain attenuation phenotypes of virusesbearing charge-cluster-to-alanine mutations in the NS5 gene of DEN4.

Table 21.

SCID-HuH-7 attenuation phenotypes of viruses bearingcharge-cluster-to-alanine mutations in the NS5 gene of DEN4.

Table 22.

Combination of paired charge-cluster-to-alanine mutations intodouble-pair mutant viruses.

Table 23.

Temperature-sensitive and mouse brain attenuation phenotypes of doublecharge-cluster-to-alanine mutants of the NS5 gene of rDEN4.

Table 24.

SCID-HuH-7 attenuation phenotypes of double charge-cluster-to-alaninemutants of the NS5 gene of rDEN4.

Table 25.

Phenotypes (temperature sensitivity, plaque size and replication inmouse brain and SCID-HuH-7 mice) of wt DEN4 and viruses containing theΔ30 and 7129 mutations.

Table 26.

The 5-fluorouracil 5-1A1 small plaque mutant demonstrates a restrictionof midgut infection following oral infection of Aedes aegyptimosquitoes.

Table 27.

The 5-fluorouracil 5-1A1 small plaque mutant demonstrates a restrictionof infection following intrathoracic inoculation of Toxorhynchitessplendens mosquitoes.

Table 28.

Mutagenesis primers for the deletion or swap of sequences in DEN4showing conserved differences from tick-borne flaviviruses.

Table 29.

Virus titer and plaque size of 3′ UTR mutant viruses in Vero and C6/36cells.

Table 30.

Infectivity of wt DEN4 and 3′ UTR mutants for Toxorhynchites splendensvia intrathoracic inoculation.

Table 31.

Infectivity of 3′ UTR swap mutant viruses for Aedes aegypti fed on aninfectious bloodmeal.

Table 32.

Putative Vero cell adaptation mutations derived from the set of 5-FUmutant viruses and other DEN4 viruses passaged in Vero cells.

Table 33.

Sequence analysis of rDEN2/4Δ30 clone 27(p4)-2-2A2.

Table 34.

Sequence analysis of rDEN2/4Δ30 clone 27(p3)-2-1A1.

Table 35.

Recombinant virus rDEN2/4Δ30 bearing Vero adaptation mutations can berecovery and titered on Vero cells.

Table 36.

Putative Vero cell adaptation mutations of dengue type 4 virus and thecorresponding wildtype amino acid residue in other dengue viruses.

Table 37.

Mutations known to attenuate dengue type 4 virus and the correspondingwildtype amino acid residue in other dengue virus.

BRIEF DESCRIPTION OF THE APPENDICES Appendix 1

Sequence of recombinant dengue type 4 virus strain 2A (amino acidsequence SEQ ID NO: 13 and nucleotide sequence SEQ ID NO: 14).

Appendix 2

Sequence of recombinant dengue type 4 virus strain rDEN4 (amino acidsequence SEQ ID NO: 15 and nucleotide sequence SEQ ID NO: 16).

Appendix 3

Sequence of recombinant dengue type 2 chimeric virus strain rDEN2/4Δ30(amino acid sequence SEQ ID NO: 17 and nucleotide sequence SEQ ID NO:18).

Appendix 4

Alignment of dengue virus polyproteins. DEN4 (SEQ ID NO: 19); DEN1-WP(SEQ ID NO: 20); DEN2-NGC (SEQ ID NO: 21); DEN3-H87 (SEQ ID NO: 22).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

To assemble a collection of useful mutations for incorporation inrecombinant live dengue virus vaccines, site-directed and randommutagenesis techniques were used to introduce mutations into the denguevirus genome. The resulting mutant viruses were screened for severalvaluable phenotypes, including temperature sensitivity in Vero cells orhuman liver cells, host cell restriction in mosquito cells or humanliver cells, host-cell adaptation for improved replication in Verocells, and attenuation in mice. The genetic basis for each observedphenotype was determined by direct sequence analysis of the virusgenome. Mutations identified through these sequencing efforts have beenfurther evaluated by their re-introduction, singly, or in combination,into recombinant dengue virus and characterization of the resultingphenotypes. In this manner, a menu of mutations was developed that isuseful in fine-tuning the attenuation and growth characteristics ofdengue virus vaccines.

Example 1 Chemical Mutagenesis of Dengue Virus Type 4 YieldsTemperature-Sensitive and Attenuated Mutant Viruses

A recombinant live attenuated dengue virus type 4 (DEN4) vaccinecandidate, 2AΔ30, was found previously to be generally well-tolerated inhumans, but a rash and an elevation of liver enzymes in the serumoccurred in some vaccinees. 2AΔ30, a non-temperature-sensitive (ts)virus, contains a 30 nucleotide deletion in the 3′ untranslated region(UTR) of the viral genome. In the present study, chemical mutagenesis ofDEN4 has been utilized to generate attenuating mutations which may beuseful to further attenuate the incompletely attenuated 2AΔ30 candidatevaccine. Wild-type DEN4 2A virus was grown in Vero cells in the presenceof 5-fluorouracil, and, from a panel of 1,248 clones that were isolatedin Vero cells, twenty ts mutant viruses were identified which were ts inboth Vero and HuH-7 cells (n=13) or in HuH-7 cells only (n=7). Each ofthe twenty ts mutations possessed an attenuation (att) phenotype asindicated by restricted replication in the brains of seven day old mice.The complete nucleotide sequence of the 20 ts mutant viruses identifiednucleotide substitutions in structural and non-structural genes as wellas in the 5′ and 3′ UTR with more than one change occurring, in general,per mutant virus. A ts mutation in the NS3 protein (nucleotide position4,995) was introduced into a recombinant DEN4 virus possessing the Δ30deletion creating the rDEN4Δ30-4995 recombinant virus which was found tobe ts and to be more attenuated than rDEN4Δ30 in the brains of mice. Amenu of attenuating mutations is being assembled that should be usefulin generating satisfactorily attenuated recombinant dengue vaccineviruses and in increasing our understanding of the pathogenesis ofdengue virus.

The mosquito-borne dengue (DEN) viruses (serotypes 1 to 4) are membersof the Flavivirus genus and contain a single-stranded positive-sense RNAgenome of approximately 10,600 nucleotides (nt) (Monath, T. P. & Heinz,F. X. 1996 in: Fields Virology B. N. Fields, et al. Eds. pp. 961-1034Lippincott-Ravan Publishers, Philadelphia). The genome organization ofDEN viruses is 5′-UTR-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3′(UTR—untranslated region, C—capsid, PrM—pre-membrane, E—envelope,NS—non-structural) (Chang, G.-J. 1997 in: Dengue and dengue hemorrhagicfever D. J. Gubler & G. Kuno, eds. pp. 175-198 CAB International, NewYork; Rice, C. M. 1996 in: Fields Virology B. N. Fields et al. Eds. pp.931-959 Lippincott-Raven Publishers, Philadelphia). A single viralpolypeptide is co-translationally processed by viral and cellularproteases generating three structural proteins (C, M, and E) and sevenNS proteins. The disease burden associated with DEN virus infection hasincreased over the past several decades in tropical and semitropicalcountries. Annually, there are an estimated 50-100 million cases ofdengue fever (DF) and 500,000 cases of the more severe and potentiallylethal dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (Gubler,D. J. & Meltzer, M. 1999 Adv Virus Res 53:35-70).

The site of viral replication in DEN virus-infected humans and thepathogenesis of DF and DHF/DSS are still incompletely understood (Innis,B. L. 1995 in: Exotic viral infections J. S. Porterfield, ed. pp.103-146 Chapman and Hall, London). In humans, DEN virus infectslymphocytes (Kurane, I. et al. 1990 Arch Virol 110:91-101;Theofilopoulos, A. N. et al. 1976 J Immunol 117:953-61), macrophages(Halstead, S. B. et al. 1977 J Exp Med 146:218-29; Scott, R. M. et al.1980 J Infect Dis 141:1-6), dendritic cells (Libraty, D. H. et al. 2001J Virol 75:3501-8; Wu, S. J. et al. 2000 Nat Med 6:816-20), andhepatocytes (Lin, Y. L. et al. 2000 J Med Virol 60:425-31; Marianneau,P. et al. 1996 J Gen Virol 77:2547-54). The liver is clearly involved inDEN virus infection of humans, as indicated by the occurrence oftransient elevations in serum alanine aminotransferase (ALT) andaspartate aminotransferase (AST) levels in the majority of denguevirus-infected patients and by the presence of hepatomegaly in somepatients (Kalayanarooj, S. et al. 1997 J Infect D is 176:313-21; Kuo, C.H. et al. 1992 Am J Trop Med Hyg 47:265-70; Mohan, B. et al. 2000 J TropPediatr 46:40-3; Wahid, S. F. et al. 2000 Southeast Asian J Trop MedPublic Health 31:259-63). DEN virus antigen-positive hepatocytes areseen surrounding areas of necrosis in the liver of fatal cases(Couvelard, A. et al. 1999 Hum Pathol 30:1106-10; Huerre, M. R. et al.2001 Virchows Arch 438:107-15), and dengue virus sequences wereidentified in such cases using RT-PCR (Rosen, L. et al. 1999 Am J TropMed Hyg 61:720-4). Of potential importance to the etiology of severedengue virus infection, three studies have demonstrated that the meanlevels of serum ALT/AST were significantly increased in patients withDHF/DSS versus those with DF (Kalayanarooj, S. et al. 1997 J Infect Dis176:313-21; Mohan, B. et al. 2000 J Trop Pediatr 46:40-3; Wahid, S. F.et al. 2000 Southeast Asian J Trop Med Public Health 31:259-63).

A vaccine for DEN viruses is not presently licensed. Since previousinfection with one dengue virus serotype can increase the risk forDHF/DSS following infection with a different serotype (Burke, D. S. etal. 1988 Am J Trop Med Hyg 38:172-80; Halstead, S. B. et al. 1969 Am JTrop Med Hyg 18:997-1021; Thein, S. et al. 1997 Am J Trop Med Hyg56:566-72), it is clear that a dengue virus vaccine will need to protectagainst each of the four dengue virus serotypes, namely DEN1, DEN2,DEN3, and DEN4. Several strategies are currently being actively pursuedin the development of a live attenuated tetravalent DEN virus vaccine(Bancroft, W. H. et al. 1984 J Infect Dis 149:1005-10; Bhamarapravati,N. & Sutee, Y. 2000 Vaccine 18:44-7; Guirakhoo, F. et al. 2000 J Virol74:5477-85; Huang, C. Y. et al. 2000 J Virol 74:3020-8). Recently, wedemonstrated that a live attenuated DEN4 vaccine candidate, 2AΔ30, wasattenuated and immunogenic in a group of 20 human volunteers (seeExample 8). This recombinant DEN4 virus contains a 30 nt deletion in the3′ UTR which removes nucleotides 10,478-10,507 and was restricted inreplication in rhesus monkeys. Levels of viremia in humans were low orundetectable, and virus recovered from the vaccinees retained the Δ30mutation. An asymptomatic rash was reported in 50% of patients. The onlylaboratory abnormality observed was an asymptomatic, transient rise inthe serum ALT level in 5 of 20 vaccinees. All vaccinees developedserum-neutralizing antibody against DEN4 virus (mean titer: 1:580).Importantly, 2AΔ30 was not transmitted to mosquitoes fed on vaccineesand has restricted growth properties in mosquitoes (Troyer, J. M. et al.2001 Am J Trop Med Hyg 65:414-9). The presence of a rash and of theelevated ALT levels suggests that the 2AΔ30 vaccine candidate isslightly under-attenuated in humans. Because of the overall set ofdesirable properties conferred by the Δ30 mutation, chimeric vaccinecandidates are being constructed which contain the structural genes ofdengue virus type 1, 2, and 3 and the DEN4 attenuated backbone bearingthe genetically stable Δ30 mutation.

Although the initial findings indicate the utility of the 2AΔ30 vaccinecandidate, many previous attempts to develop live attenuated denguevirus vaccines have yielded vaccine candidates that were either over- orunder-attenuated in humans (Eckels, K. H. et al. 1984 Am J Trop Med Hyg33:684-9; Bhamarapravati, N. & Yoksan, S. 1997 in: Dengue and denguehemorrhagic fever D. J. Gubler & G. Kuno eds. pp. 367-377 CABInternational, New York; Innis, B. L. et al. 1988 J Infect Dis158:876-80; McKee, K. T., Jr. et al. 1987 Am J Trop Med Hyg 36:435-42).Therefore, we developed a menu of point mutations which confertemperature-sensitive (ts) and attenuation (att) phenotypes upon DEN4.These mutations are envisioned as being useful to attenuate DEN4 virusesto different degrees and therefore as having purpose in fine-tuning thelevel of attenuation of vaccine candidates such as 2AΔ30. Addition ofsuch mutations to 2AΔ30 or to other dengue virus vaccine candidates isenvisioned as resulting in the generation of a vaccine candidate thatexhibits a satisfactory balance between attenuation and immunogenicityfor humans.

In the present example, chemical mutagenesis of DEN4 has been utilizedto identify point mutations which confer the ts phenotype, since suchviruses often are attenuated in humans. Additionally, because of thereported involvement of the liver in natural dengue infection and theelevated ALT levels in a subset of 2AΔ30 vaccinees, mutagenized DEN4viruses were also evaluated for ts phenotype in HuH-7 liver cellsderived from a human hepatoma. Here, we describe the identification of20 DEN4 ts mutant viruses each of which replicates efficiently in Verocells, the proposed substrate for vaccine manufacture, and each of whichis attenuated in mice. Finally, the feasibility of modifying theattenuation phenotype of the 2AΔ30 vaccine candidate by introduction ofa point mutation in NS3 is demonstrated.

Cells and Viruses.

WHO Vero cells (African green monkey kidney cells) were maintained inMEM (Life Technologies, Grand Island, N.Y.) supplemented with 10% fetalbovine serum (FBS) (Summit Biotechnologies, Fort Collins, Colo.), 2 mML-glutamine (Life Technologies), and 0.05 mg/ml gentamicin (LifeTechnologies). HuH-7 cells (human hepatoma cells) (Nakabayashi, H. etal. 1982 Cancer Res 42:3858-63) were maintained in D-MEM/F-12 (LifeTechnologies) supplemented with 10% FBS, 1 mM L-glutamine and 0.05 mg/mlgentamicin. C6/36 cells (Aedes albopictus mosquito cells) weremaintained in complete MEM as described above supplemented with 2 mMnon-essential amino acids (Life Technologies).

The wild type (wt) DEN4 2A virus was derived from a cDNA clone of DEN4strain 814669 (Dominica, 1981) (Men, R. et al. 1996 J Virol 70:3930-7).Sequence of the cDNA of DEN 4 2A virus is presented in Appendix 1. Thefull-length 2A cDNA clone has undergone several subsequent modificationsto improve its ability to be genetically manipulated. As previouslydescribed, a translationally-silent XhoI restriction enzyme site wasengineered near the end of the E region at nucleotide 2348 to createclone 2A-XhoI (Bray, M. & Lai, C. J. 1991 PNAS USA 88:10342-6). Theviral coding sequence of the 2A-XhoI cDNA clone was further modifiedusing site-directed mutagenesis to create clone p4: a unique BbvCIrestriction site was introduced near the C-prM junction (nucleotides447-452); an extra XbaI restriction site was ablated by mutation ofnucleotide 7730; and a unique SacII restriction site was created in theNS5 region (nucleotides 9318-9320). Each of these engineered mutationsis translationally silent and does not change the amino acid sequence ofthe viral polypeptide. Also, several mutations were made in the vectorregion of clone p4 to introduce or ablate additional restriction sites.The cDNA clone p4Δ30 was generated by introducing the Δ30 mutation intoclone p4. This was accomplished by replacing the MluI-KpnI fragment ofp4 (nucleotides 10403-10654) with that derived from plasmid 2AΔ30containing the 30 nucleotide deletion. The cDNA clones p4 and p4Δ30 weresubsequently used to generate recombinant viruses rDEN4 (Appendix 2) andrDEN4α30, respectively. (The GenBank accession number for rDEN4 isAF326825 and the accession for rDEN4Δ30 is AF326827).

Chemical Mutagenesis of DEN4.

Confluent monolayers of Vero cells were infected with wt DEN4 2A at anmultiplicity of infection (MOI) of 0.01 and incubated for 2 hours at 32°C. Infected cells were then overlaid with MEM supplemented with 2% FBSand 5-fluorouracil (5-FU) (Sigma, St. Louis, Mo.) at concentrationsranging from 10 mM to 10 nM. After incubation at 32° C. for five days,cell culture medium was harvested, clarified by centrifugation, andfrozen at −70° C. Clarified supernatants were then assayed for virustiter by plaque titration in Vero cells. Serial ten-fold dilutions ofthe clarified supernatant were prepared in Opti-MEM I (LifeTechnologies) and inoculated onto confluent Vero cell monolayers in24-well plates. After incubation at 35° C. for two hours, monolayerswere overlaid with 0.8% methylcellulose (EM Science, Gibbstown, N.J.) inOpti-MEM I supplemented with 2% FBS, gentamicin, and L-glutamine.Following incubation at 35° C. for five days, plaques were visualized byimmunoperoxidase staining. Vero cell monolayers were fixed in 80%methanol for 30 minutes and washed for 10 minutes with antibody bufferwhich consists of 3.5% (w/v) nonfat dry milk (Nestle, Solon, Ohio) inphosphate buffered saline (PBS). Cells were then incubated for one hourat 37° C. with an anti-DEN4 rabbit polyclonal antibody preparation(PRNT₅₀ of >1:2000) diluted 1:1,000 in antibody buffer. After one washwith antibody buffer, cells were incubated for one hour withperoxidase-labeled goat-anti-rabbit IgG (KPL, Gaithersburg, Md.) diluted1:500 in antibody buffer. Monolayers were washed with PBS, allowed todry briefly, overlaid with peroxidase substrate (KPL), and plaques werecounted.

Virus yields in cultures treated with 1 mM 5-FU were reduced 100-foldcompared to untreated cultures, and the virus present in the supernatantfrom the 1 mM 5-FU-treated culture was terminally diluted to deriveclones for phenotypic characterization. Briefly, 96 well plates of Verocells were inoculated with the 5-FU-treated virus at an MOI that yielded10 or fewer virus-positive wells per plate. After a five-day incubationat 35° C., cell culture media from the 96 well plates were temporarilytransferred to 96 well plates lacking cells, and the positive cultureswere identified by immunoperoxidase staining of the infected-cellmonolayers. Virus from each positive well was transferred to confluentVero cell monolayers in 12 well plates for amplification. Cell culturemedium was harvested from individual wells five or six days later,clarified by centrifugation, aliquoted to 96 deep-well polypropyleneplates (Beckman, Fullerton, Calif.) and frozen at −70° C. A total of1,248 virus clones were prepared from the 1 mM 5-FU-treated cultures.Two wt virus clones, 2A-1 and 2A-13, were generated in the same mannerfrom the 5-FU untreated control cultures.

Screening of Clones for Ts and Att Phenotypes.

The 1,248 virus clones were screened for ts phenotype by assessing virusreplication at 35° C. and 39° C. in Vero and HuH-7 cells. Cellmonolayers in 96 well plates were inoculated with serial ten-folddilutions of virus in L-15 media (Quality Biologicals, Gaithersburg,Md.) supplemented with 2% FBS, L-glutamine and gentamicin. Cells wereincubated at the indicated temperatures for five days intemperature-controlled water baths, and presence of virus was determinedby immunoperoxidase staining as described above. Virus clones whichdemonstrated a 100-fold or greater reduction in titer at 39° C. wereterminally diluted an additional two times and amplified in Vero cells.The efficiency of plaque formation (EOP) at permissive and restrictivetemperatures of each triply biologically cloned virus suspension wasdetermined as follows. Plaque titration in Vero and HuH-7 cells wasperformed as described above except virus-infected monolayers wereoverlaid with 0.8% methylcellulose in L-15 medium supplemented with 5%FBS, gentamicin, and L-glutamine. After incubation of replicate platesfor five days at 35, 37, 38, or 39° C. in temperature-controlled waterbaths, plaques were visualized by immunoperoxidase staining and counted.

The replication of DEN4 5-FU ts mutant viruses was evaluated in SwissWebster suckling mice (Taconic Farms, Germantown, N.Y.). Groups of sixone-week-old mice were inoculated intracranially with 10⁴ PFU of virusdiluted in 30 μl Opti-MEM I. Five days later, mice were sacrificed andbrains were removed and individually homogenized in a 10% suspension ofphosphate-buffered Hank's balanced salt solution containing 7.5%sucrose, 5 mM sodium glutamate, 0.05 mg/ml ciprofloxacin, 0.06 mg/mlclindamycin, and 0.0025 mg/ml amphotericin B. Clarified supernatantswere frozen at −70° C. and subsequently virus titer was determined bytitration in Vero cells, and plaques were stained by theimmunoperoxidase method described above.

Sequence Analysis of Viral Genomes.

The nucleotide sequence of the 5-FU-mutagenized DEN4 viruses wasdetermined. Briefly, genomic viral RNA was isolated from virus cloneswith the QIAamp viral RNA mini kit (Qiagen, Valencia, Calif.) andreverse transcription was performed using the SuperScript First StrandSynthesis System for RT-PCR (Life Technologies) and random hexamerprimers. Advantage cDNA polymerase (Clontech, Palo Alto, Calif.) wasused to generate overlapping PCR fragments of approximately 2,000 ntwhich were purified by HighPure PCR Product Purification System (RocheDiagnostics, Indianapolis, Ind.). DEN-specific primers were used inBigDye terminator cycle sequencing reactions (Applied Biosystems, FosterCity, Calif.) and reactions were analyzed on a 3100 genetic analyzer(Applied Biosystems). Primers were designed to sequence both strands ofthe PCR product from which consensus sequences were assembled.

The nucleotide sequence of the 5′ and 3′ regions of the viral genomewere determined as above after circularization of the RNA genome. The 5′cap nucleoside of the viral RNA was excised using tobacco acidpyrophosphatase (Epicentre Technologies, Madison, Wis.) and the genomewas circularized by RNA ligase (Epicentre Technologies). A RT-PCRfragment was generated which overlapped the ligation junction (5′ and 3′ends) and was sequenced as described above.

Generation of Recombinant DEN4 Viruses.

The mutation at nt position 4,995 in NS3 was introduced into the p4 cDNAconstruct by site-directed mutagenesis (Kunkel, T. A. 1985 PNAS USA82:488-92). The StuI-BstBI (nt 3,619-5,072) fragment of p4 wassub-cloned into a modified pUC119 vector. The U>C mutation at ntposition 4,995 was engineered by site-directed mutagenesis into the p4fragment, cloned back into the p4 cDNA construct, and the presence ofthe mutation was confirmed by sequence analysis. The Δ30 mutation wasintroduced into the 3′ UTR of the p4-4995 cDNA clone by replacing theMluI-KpnI fragment with that derived from the p4Δ30 cDNA clone, and thepresence of the deletion was confirmed by sequence analysis. Full lengthRNA transcripts were prepared from the above cDNA clones by in vitrotranscription. Briefly, transcription consisted of a 50 μA reactionmixture containing 1 μg linearized plasmid, 60 U SP6 polymerase (NewEngland Biolabs (NEB), Beverly, Mass.), 1×RNA polymerase buffer (40 mMTris-HCl, pH 7.9, 6 mM MgCl₂, 2 mM spermidine, 10 mM dithiothreitol),0.5 mM m7G(5′)ppp(5′)G cap analog (NEB), 1 mM each nucleotidetriphosphate, 1 U pyrophosphatase (NEB), and 80 U RNAse inhibitor(Roche, Indianapolis, Ind.). This reaction mixture was incubated at 40°C. for 90 min and the resulting transcripts were purified using RNeasymini kit (Qiagen, Valencia, Calif.).

For transfection of C6/36 cells, RNA transcripts were combined withDOTAP liposomal transfection reagent (Roche) in HEPES-buffered saline(pH 7.6) and added to cell monolayers in 6 well plates. After incubationat 32° C. for 12-18 hours, the cell culture media were removed andreplaced with MEM supplemented with 5% FBS, L-glutamine, gentamicin andnon-essential amino acids. Cell monolayers were incubated for anadditional 5 to 7 days and cell culture media were harvested, clarifiedby centrifugation, and assayed for the presence of virus by plaquetitration in Vero cells. Recovered viruses were terminally diluted twiceas described above, and virus suspensions for further analysis wereprepared in Vero cells.

In Vitro (Tissue Culture) and In Vivo Replication of Wt DEN4 andDEN4Δ30.

The level of replication of both wt DEN4 2A and the vaccine candidate,2AΔ30, was evaluated in Vero (monkey kidney) and HuH-7 (human hepatoma)cells (FIG. 1), the latter of which has recently been found toefficiently support the replication of DEN2 virus (Lin, Y. L. et al.2000 J Med Virol 60:425-31). The pattern of replication of wt DEN4 2Aand 2AΔ30 was similar in both cell lines. Viral titers from culturesinfected with 2AΔ30 at an MOI of 0.01 were slightly reduced compared towt DEN4 2A at 72 hours, but at later time points their level ofreplication was equivalent. The efficient replication of both DEN4viruses in each cell line indicated that these continuous lines of cellswould be useful for characterization of the is phenotype of the 1248potential mutant viruses.

The level of replication of DEN4 virus administered intracerebrally toSwiss Webster mice was first determined to assess whether mice could beused to efficiently evaluate and quantitate the attenuation phenotype ofa large set of mutant viruses. Since the susceptibility of mice to DENinfection is age dependent (Cole, G. A. & Wisseman, 1969 Am J Epidemiol89:669-80; Cole, G. A. et al. 1973 J Comp Pathol 83:243-52), mice aged 7to 21 days were infected with 2A-13 (a clone of DEN4 wild type virus—seebelow), rDEN4 or rDEN4Δ30, and after five days the brain of each mousewas removed, and the level of viral replication was quantitated byplaque assay (Table 1). The results indicated that the two wt DEN4viruses and the rDEN4Δ30 vaccine candidate replicated to high titer(>6.0 log₁₀ PFU/g brain) in 7-day old mice and that the mean viraltiters were similar among the three viruses. These results demonstratedthe feasibility of using 7-day old mice to screen a large set of mutantviruses, and the high level of replication of wild type and vaccinecandidate permits one to quantitate the magnitude of the restriction ofreplication specified by an attenuating mutation over a 10,000-foldrange.

Generation and In Vitro Characterization of DEN4 5-FU Mutant viruses.

A panel of 1,248 DEN4 virus clones was generated from a 5-FU-mutagenizedsuspension of wt DEN4 2A as described above (FIG. 2). Each clone wastested in Vero and HuH-7 cells for the ts phenotype at 39° C., andputative ts mutant viruses were subjected to two additional rounds ofbiological cloning by terminal dilution, and the ts phenotype of eachfurther cloned virus population was examined in more detail bydetermining their efficiency of plating (EOP) at permissive temperature(35° C.) and at various restrictive temperatures (Table 2). One virus(clone 2A-13) without a ts phenotype, which was passaged in an identicalfashion as the ts mutant viruses, served as the virus to which each ofthe ts mutant viruses was directly compared for both the ts and attphenotypes.

Thirteen 5-FU mutant viruses were identified which have a ts phenotypein both Vero and HuH-7 cells, and seven mutant viruses were ts only inHuH-7 cells (Table 2). Mutant viruses which were ts in Vero cells butnot in HuH-7 cells were not identified. Temperature-sensitivity wasdefined as a ≧2.5 or ≧3.5 log₁₀ PFU/ml reduction in virus titer in Veroor HuH-7 cells, respectively, at an indicated temperature when comparedto the permissive temperature of 35° C. Wild type DEN4 2A was found tohave approximately a 0.5 and 1.5 log₁₀ PFU/ml reduction in virus titerin Vero or HuH-7 cells at 39° C., respectively. The Δ30 deletion did notconfer a ts phenotype in Vero or HuH-7 cells and exhibited only a slightreduction in virus titer (2.2 log₁₀ PFU/ml) at 39° C. in HuH-7 cells,which was less than 10-fold greater than the reduction of wt DEN4 2A atthat temperature. Several 5-FU mutant viruses had a greater than10,000-fold reduction in virus titer at 39° C. in both Vero and HuH-7cells. A complete shut-off in viral replication at 39° C. in HuH-7 cellswas observed in five virus clones (#571, 605, 631, 967, and 992) whichwere not ts in Vero cells. Mutations that selectively restrictreplication in HuH-7 liver cells may be particularly useful incontrolling the replication of dengue virus vaccine candidates in theliver of vaccinees.

Replication of DEN4 5-FU Mutant Viruses in Suckling Mice.

The level of replication of each of the 20 ts DEN4 mutant viruses inmouse brain was determined (Table 2). The titers obtained were comparedto that of the two wt viruses, 2A-13 and rDEN4, which each replicated toa level of greater than 10⁶ PFU/g of brain tissue, and to that of the2AΔ30 mutant, which conferred only a limited 0.5 log₁₀ PFU/g reductionin mean virus titer compared to the wt controls. The observed reductionin the level of rDEN4Δ30 replication was consistent among 11 separateexperiments. Interestingly, the rDEN4Δ30 virus, which was attenuated inboth rhesus monkeys and humans (Example 8), was only slightly restrictedin replication in mouse brain. Varying levels of restriction ofreplication were observed among the mutant viruses ranging from a10-fold (#473) to over 6,000-fold (#686) reduction. Mutant viruses withts phenotypes in both Vero and HuH-7 cells, as well as in HuH-7 cellsalone, were found to have significant att phenotypes. Five of 13 5-FUmutant viruses with ts phenotypes in both Vero and HuH-7 cells and fiveof seven mutant viruses with ts phenotypes in HuH-7 cells alone hadgreater than a 100-fold reduction in virus replication. There appearedto be no direct correlation between the magnitude of the reduction inreplication at restrictive temperature in tissue culture and the levelof attenuation in vivo. The similar level of temperature sensitivity andreplication of the rDEN4 wt and clone 2A-13 in mouse brain indicatedthat observed differences in replication between the ts mutant virusesand clone 2A-13 was not simply a function of passage in Vero cells, butreflects the sequence differences between these viruses.

Sequence Analysis of DEN4 5-FU Mutant Viruses.

To determine the genetic basis of the observed ts and att phenotypes,the complete nucleotide sequence of each ts mutant and of clone 2A-13was determined and summarized in Table 3 (ts in Vero and HuH-7 cells)and Table 4 (ts in only HuH-7 cells).

The only type of mutation identified in the 20 mutant viruses sequencedwas a nucleotide substitution (no deletions or insertions occurred), andthese were present in each of the coding regions except C and NS4A.Three mutant viruses (#239, 489, and 773) contained only a singlemissense point mutation in NS3 at nt position 4,995 resulting in a Serto Pro amino acid (a.a.) change at a.a. position 1,632. For #773, thiswas the sole mutation present (Table 3). The non-coding mutations incoding regions are not considered to be significant. The 17 additionalmutant viruses had multiple mutations (two to five) in a coding regionor in an UTR which could potentially confer the observed ts or attphenotypes. Five of the 17 mutant viruses with multiple mutations (#473,718, 759, 816, and 938) also encoded the point mutation at nt position4,995. The presence of the 4,995 mutation was found in only DEN4 mutantviruses with ts phenotypes in both Vero and HuH-7 cells.

The sequence analysis indicated that 10 mutant viruses which were ts inVero and HuH-7 cells and three mutant viruses which were ts in onlyHuH-7 cells contained mutations in only the 5′ and 3′ UTR and/or in anonstructural protein. These mutations are especially suitable forinclusion in chimeric dengue virus vaccine candidates in which thestructural genes derive from a DEN1, DEN2, or DEN3 serotype and theremaining coding and non-coding regions come from an attenuated DEN4vector. Mutations identified in 5-FU DEN4 mutant viruses which were tsin only HuH-7 cells (Table 4) may potentially be utilized in vaccinecandidates, such as rDEN4Δ30, to selectively control the replication andpathogenesis of DEN4 in the liver. These combined results from thesequence analysis of 5-FU mutant viruses demonstrate the utility ofchemical mutagenesis as a means of introducing attenuating mutationsinto the dengue virus genome.

The presence of a point mutation at nt position 4,995 in eight separatemutant viruses was described above. Five additional point mutations werealso represented in multiple viruses including nt changes at position1,455 in E, 7,162, 7,163 and 7,564 in NS4B, and 10,275 in the 3′ UTR(Table 5). The significance of the occurrence of these “sister”mutations in multiple viruses is discussed in Example 6. Interestingly,the wild-type, parallel-passaged virus, 2A-13, also contained a singlemutation at the 7,163 nt position in NS4B.

Introduction of a is Mutation into rDEN4 and rDEN4Δ30.

The presence of a single nucleotide substitution (U>C mutation at ntposition 4,995 in NS3) in three separate mutant viruses (clones 239,489, and 773) indicated that this mutation specified the ts and attphenotypes in each of the three mutant viruses. This mutation was clonedinto cDNA construct of p4 and p4Δ30 and recombinant viruses wererecovered and designated rDEN4-4995 and rDEN4Δ30-4995, respectively.These recombinant viruses were tested for ts and att phenotypes asdescribed above (Table 6). As expected, introduction of mutation 4995into rDEN4 wt resulted in a significant ts phenotype at 39° C. in bothVero and HuH-7 cells. rDEN4-4995 grew to nearly wild-type levels at thepermissive temperature, 35° C., in both cell types, but demonstrated agreater than 10,000-fold reduction at 39° C. (shut-off temperature) inboth Vero and HuH-7 cells. The addition of the 4995 mutation to rDEN4Δ30yields a recombinant virus, rDEN4Δ30-4995, that exhibits the same levelof temperature sensitivity as rDEN4-4995 (Table 6).

The rDEN4 viruses encoding the 4995 mutation were next tested forreplication in the brains of suckling mice (Table 6). The 4995 mutationconferred an att phenotype upon both rDEN4 and rDEN4Δ30. There was anapproximately 1,000-fold reduction in virus replication compared to thatof wt virus. The combination of point mutation 4995 and the Δ30 deletiondid not appear to result in an additive reduction of virus replication.These results confirmed that the 4995 point mutation indeed specifiesthe ts and att phenotypes. Importantly, the utility of modifying tissueculture and in vivo phenotypes of the rDEN4Δ30 vaccine candidate byintroduction of additional mutations was also demonstrated.

Discussion.

Herein we teach how to prepare a tetravalent, live-attenuated denguevirus vaccine using rDEN4Δ30 as the DEN4 component and three antigenicchimeric viruses expressing the structural proteins (C, prM, and E) ofDEN1, DEN2, and DEN3 from the attenuated rDEN4Δ30 vector (Example 8).DEN4 virus rDEN4Δ30 containing the Δ30 deletion mutation in the 3′ UTRmanifests restricted replication in humans while retainingimmunogenicity. Since rDEN4Δ30 retains a low level of residual virulencefor humans despite this restricted replication, the present study wasinitiated to generate additional attenuating mutations that areenvisioned as being useful to further attenuate rDEN4Δ30 or other dengueviruses and that are envisioned as being incorporated into any of thethree antigenic chimeric viruses or other dengue viruses as needed.Temperature-sensitive mutants of dengue viruses (Bhamarapravati, N. &Yoksan, S. 1997 in: Dengue and Dengue Hemorrhagic Fever D. J. Gubler &G. Kuno eds. pp. 367-377 CAB International, New York; Eckels, K. H. etal. 1980 Infect Immun 27:175-80) as well of other viruses (Skiadopoulos,M. H. et al. 1998 J Virol 72:1762-8; Whitehead, S. S. et al. 1999 JVirol 73:871-7) manifest restricted replication in vivo. We havegenerated a panel of 20 ts DEN4 mutant viruses, determined their genomicsequence, and assessed their in vivo attenuation phenotypes. The 20 tsDEN4 mutant viruses were generated by growth in the presence of 5-FU andwere first selected for viability in Vero cells, the substrate plannedfor use in the manufacture of these vaccines, to ensure that the mutantviruses can be grown efficiently in a suitable substrate.

Two classes of mutant viruses were obtained; those ts in both Vero andHuH-7 cells (n=13) or those ts in only HuH-7 cells (n=7). The virusesexhibited a range in their level of temperature sensitivity from a 100-to 1,000,000-fold reduction in replication at the restrictivetemperature of 39° C. Since our DEN4 vaccine candidate retains a lowlevel of virulence for the liver and other findings support the abilityof dengue viruses to infect hepatocytes (Lin, Y. L. et al. 2000 J MedVirol 60:425-31; Marianneau, P. et al. 1997 J Virol 71:3244-9) and causeliver pathology (Couvelard, A. et al. 1999 Hum Pathol 30:1106-10;Huerre, M. R. et al. 2001 Virchows Arch 438:107-15), we sought todevelop mutations that would selectively restrict replication of dengue4 virus in liver cells. Toward this end, we identified seven mutantviruses which have a HuH-7 cell-specific ts phenotype. The mutationspresent in these viruses are the first reported in DEN viruses thatconfer restricted replication in liver cells and are envisioned as beinguseful in limiting virus replication and pathogenesis in the liver ofvaccine recipients. The contribution of individual mutations identifiedin the HuH-7 cell-specific ts viruses to the observed phenotypes isenvisioned as being assessed by introduction of the individual mutationsinto recombinant DEN4 viruses.

Recent evidence has indicated that the magnitude of the viremia inDEN-infected patients positively correlates with disease severity, i.e.,the higher the titer of viremia the more severe the disease (Murgue, B.et al. 2000 J Med Virol 60:432-8; Vaughn, D. W. et al. 2000 J Infect Dis181:2-9). This indicates that mutations that significantly restrictreplication of vaccine candidates in vivo are the foundation of a safeand attenuated vaccine. Evaluation of DEN virus vaccine candidates forin vivo attenuation is complicated by the lack of a suitable animalmodel which accurately mimics the disease caused by dengue viruses inhumans. In the absence of such a model, the replication of the panel of5-FU mutant viruses in the brains of Swiss Webster suckling mice wasassessed as a means to identify an in vivo attenuation phenotype sincethis animal model is well-suited for the evaluation of a large set ofmutant viruses. Each of the 20 ts mutant viruses exhibited an attphenotype manifesting a 10- to 6,000-fold reduction in replication inthe brain of mice as compared to wt DEN4 virus (Table 2). This indicatesthat there is a correlation between the presence of the ts phenotype intissue culture and attenuation of the mutant in vivo confirming theutility of selecting viruses with this marker as vaccine candidates.However, there was no correlation between the level of temperaturesensitivity and the level of restriction in vivo. Furthermore, Sabinobserved a dissociation between mouse neurovirulence and attenuation inhumans by generating an effective live attenuated virus vaccine againstDEN by passage of virus in mouse brain. This research actually resultedin a highly mouse-neurotropic DEN virus which, paradoxically, wassignificantly attenuated in humans (Sabin, A. B. 1952 Am J Trop Med Hyg1:30-50). Despite this, attenuation for the suckling mouse brain hasbeen reported for other live-attenuated DEN virus vaccine candidatesincluding the DEN2 PDK-53 vaccine strain which is non-lethal in mice andDEN-2 PR-159/S-1 vaccine strain which was significantly attenuatedcompared to its parental wild-type virus (Bhamarapravati, N. & Yoksan,S. 1997 in: Dengue and Dengue Hemorrhagic Fever D. J. Gubler & G. Kunoeds. pp. 367-377 CAB International, New York; Butrapet, S. et al. 2000 JVirol 74:3011-9; Eckels, K. H. et al. 1980 Infect Immun 27:175-80;Innis, B. L. et al. 1988 J Infect Dis 158:876-80). Replication in rhesusmonkeys has been reported to be predictive of attenuation for humans(Innis, B. L. et al. 1988 J Infect Dis 158:876-80). Recently, murinemodels of DEN virus infection have been developed using SCID micetransplanted with human macrophage (Lin, Y. L. et al. 1998 J Virol72:9729-37) or liver cell lines (An, J. et al. 1999 Virology 263:70-7),but these mice have not as yet been used to assess att phenotypes ofcandidate vaccine viruses. Mutant viruses or recombinant viruses bearingone or more of these mutations described herein are envisioned as beingtested for replication in rhesus monkeys (or other suitable animalmodel) as predictive for attenuation in humans.

The chemical mutagenesis of DEN4 virus and sequence analysis ofresulting viruses described here has resulted in the identification of alarge number of point mutations resulting in amino acid substitutions inall genes except C and NS4A as well as point mutations in the 5′ and 3′UTR (Tables 3 and 4). This approach of whole-genome mutagenesis has thebenefit of identifying mutations dispersed throughout the entire genomewhich are pre-selected for viability in the Vero cell substrate. Ten5-FU mutant viruses which were ts in Vero and HuH-7 cells and threeviruses which were selectively ts in HuH-7 cells contained onlymutations outside of the genes encoding the structural proteins, i.e.,in the 5′ and 3′ UTR or NS genes. These mutations along with the Δ30deletion in the 3′ UTR are particularly suited for inclusion inantigenic, chimeric vaccines which consist of an attenuated DEN4 vectorbearing the wild-type structural genes (C, prM, E) of the other DENvirus serotypes. Use of this strategy has several advantages. Eachantigenic chimeric virus that possesses structural proteins from awild-type virus along with attenuating mutations in their UTRs or NSgenes should maintain its infectivity for humans, which is mediatedlargely by the E protein, and, therefore, each vaccine component shouldbe immunogenic (Huang, C. Y. et al. 2000 J Virol 74:3020-8). Thereplicative machinery of the tetravalent vaccine strains would share thesame attenuating mutations in the NS genes or in the UTR which shouldattenuate each vaccine component to a similar degree and therebyminimize interference or complementation among the four vaccine viruses.In addition, wild-type E protein would be expected to most efficientlyinduce neutralizing antibodies against each individual DEN virus.

Sequence analysis of dengue viruses (Blok, J. et al. 1992 Virology187:573-90; Lee, E. et al. 1997 Virology 232:281-90; Puri, B. et al.1997 J Gen Virol 78:2287-91) and yellow fever viruses (Dunster, L. M. etal. 1999 Virology 261:309-18; Holbrook, M. R. et al. 2000 Virus Res69:31-9) previously generated by serial passage in tissue culture havemutations throughout much of the genome, a pattern we have observed inthe present study. Recent analysis of the DEN2 PDK-53 vaccine strain hasidentified the important mutations involved in attenuation which werelocated in non-structural regions including the 5′ UTR, NS1 and NS3(Butrapet, S. et al. 2000 J Virol 74:3011-9). This DEN2 vaccine strainhas been used to generate a chimeric virus with DEN1 C-prM-E genes(Huang, C. Y. et al. 2000 J Virol 74:3020-8). In separate studies, thesequence of the DEN1 vaccine strain 45AZ5 PDK-27 was determined andcompared to parental viruses, but the mutations responsible forattenuation have not yet been identified (Puri, B. et al. 1997 J GenVirol 78:2287-91).

Several amino acid substitutions were identified in more than one is5-FU mutant virus (Table 5). Lee et al. have previously reported findingrepeated mutations in separate DEN3 virus clones after serial passage inVero cells (Lee, E. et al. 1997 Virology 232:281-90). A mutation (K>N)identified in E at a.a. position 202 in a single DEN3 passage series wasalso found in our 5-FU mutant virus #1012 (K>E). Mutations observed inthe 5-FU sister mutant viruses are envisioned as representing adaptivechanges that confer an increased efficiency of DEN4 replication in Verocells. Such mutations are envisioned as being beneficial for inclusionin a live-attenuated DEN virus vaccine by increasing the yield ofvaccine virus during manufacture. Interestingly, three distinct aminoacid substitutions were found in NS4B of the 5-FU sister mutant viruses.The exact function of this gene is unknown, but previous studies oflive-attenuated yellow fever vaccines (Jennings, A. D. et al. 1994 JInfect Dis 169:512-8; Wang, E. et al. 1995 J Gen Virol 76:2749-55) andJapanese encephalitis vaccines (Ni, H. et al. 1995 J Gen Virol76:409-13) have identified mutations in NS4B associated with attenuationphenotypes.

The mutation at nt position 4995 of NS3 (S1632P) was present as the onlysignificant mutation identified in three 5-FU mutant viruses (#239,#489, and #773). This mutation was introduced into a recombinant DEN4virus and found to confer a ts and att phenotype (Table 6). Theseobservations clearly identify the 4995 mutation as an attenuatingmutation. Analysis of a sequence alignment (Chang, G.-J. 1997 in: Dengueand Dengue Hemorrhagic Fever D. J. Gubler & G. Kuno, eds. pp. 175-198CAB International, New York) of the four dengue viruses indicated thatthe Ser at a.a. position 1632 is conserved in DEN1 and DEN2, while DEN3contains an Asn at this position indicating that the mutation ispredicted to be useful in modifying the phenotypes of the other DENvirus serotypes. The NS3 protein is 618 a.a. in length and contains bothserine protease and helicase activities (Bazan, J. F. & Fletterick, R.J. 1989 Virology 171:637-9; Brinkworth, R. I. et al. 1999 J Gen Virol80:1167-77; Valle, R. P. & Falgout, B. 1998 J Virol 72:624-32). The 4995mutation results in a change at a.a. position 158 in NS3 which islocated in the N-terminal region containing the protease domain. Aminoacid position 158 is located two a.a. residues away from an NS3conserved region designated homology box four. This domain has beenidentified in members of the flavivirus family and is believed to be acritical determinant of the NS3 protease substrate specificity (Bazan,J. F. & Fletterick, R. J. 1989 Virology 171:637-9; Brinkworth, R. I. etal. 1999 J Gen Virol 80:1167-77). However, the exact mechanism whichresults in the phenotype associated with the 4995 mutation has not yetbeen identified. The identification of the 4995 mutation as anattenuating mutation permits a prediction of its usefulness for thefurther attenuation of rDEN4Δ30.

We have determined the contribution of individual 5-FU mutations to theobserved phenotypes by introduction of the mutations into recombinantDEN4 viruses as was demonstrated herein for the 4995 mutation (seeExample 3). In addition, combination of individual mutations with eachother or with the Δ30 mutation is useful to further modify theattenuation phenotype of DEN4 virus candidate vaccines. The introductionof the 4995 mutation into rDEN4Δ30 described herein rendered therDEN4Δ30-4995 double mutant ts and 1000-fold more attenuated for themouse brain than rDEN4Δ30. This observation has demonstrated thefeasibility of modifying both tissue culture and in vivo phenotypes ofthis and other dengue virus vaccine candidates. Once the mutationsresponsible for the HuH-7 cell-specific ts phenotype are identified asdescribed above and introduced into the rDEN4Δ30 vaccine candidate, weenvision confirming that these mutations attenuate rDEN4Δ30 vaccinevirus for the liver of humans. A menu of attenuating mutations isenvisioned as being assembled that is predicted to be useful ingenerating satisfactorily attenuated recombinant dengue vaccine virusesand in increasing our understanding of the pathogenesis of dengue virus(see Example 7).

Example 2 Chemical Mutagenesis of DEN4 Virus Results in Small-PlaqueMutant Viruses with Temperature-Sensitive and Attenuation Phenotypes

Mutations that restrict replication of dengue virus have been sought forthe generation of recombinant live-attenuated dengue virus vaccines.Dengue virus type 4 (DEN4) was previously grown in Vero cells in thepresence of 5-fluorouracil, and the characterization of 1,248mutagenized, Vero cell-passaged clones identified 20temperature-sensitive (ts) mutant viruses that were attenuated (att) insuckling mouse brain (Example 1). The present investigation has extendedthese studies by identifying an additional 22 DEN4 mutant viruses whichhave a small-plaque size (sp) phenotype in Vero cells and/or the livercell line, HuH-7. Five mutant viruses have a sp phenotype in both Veroand HuH-7 cells, three of which are also ts. Seventeen mutant viruseshave a sp phenotype in only HuH-7 cells, thirteen of which are also ts.Each of the sp viruses was growth restricted in the suckling mousebrain, exhibiting a wide range of reduction in replication (9- to100,000-fold). Complete nucleotide sequence was determined for the 22DEN4 sp mutant viruses, and nucleotide substitutions were found in the3′ untranslated region (UTR) as well as in all coding regions exceptNS4A. Identical mutations have been identified in multiple virus clonesindicating that they are involved in the adaptation of DEN4 virus toefficient growth in Vero cells.

The DEN viruses cause more disease and death of humans than any otherarbovirus, and more than 2.5 billion people live in regions with endemicdengue infection (Gubler, D. J. 1998 Clin Microbiol Rev 11:480-96).Annually, there are an estimated 50-100 million cases of dengue fever(DF) and 500,000 cases of the more severe and potentially lethal denguehemorrhagic fever/dengue shock syndrome (DHF/DSS) (Gubler, D. J. &Meltzer, M. 1999 Adv Virus Res 53:35-70). Dengue fever is an acuteinfection characterized by fever, retro-orbital headache, myalgia, andrash. At the time of defervescence during DF, a more severe complicationof DEN virus infection, DHF/DSS, may occur which is characterized by asecond febrile period, hemorrhagic manifestations, hepatomegaly,thrombocytopenia, and hemoconcentration, which may lead to potentiallylife-threatening shock (Gubler, D. J. 1998 Clin Microbiol Rev11:480-96).

The sites of DEN virus replication in humans and their importance andrelationship to the pathogenesis of DF and DHF/DSS are stillincompletely understood (Innis, B. L. 1995 in: Exotic Viral InfectionsJ. S. Porterfield, ed. pp. 103-146 Chapman and Hall, London). Inaddition to replication in lymphoid cells, it has become evident thatthe liver is involved in DEN infection of humans. Transient elevationsin serum alanine aminotransferase (ALT) and aspartate aminotransferase(AST) levels are observed in the majority of DEN virus-infected patientsand hepatomegaly is observed in some patients (Kalayanarooj, S. et al.1997 J Infect Dis 176:313-21; Kuo, C. H. et al. 1992 Am J Trop Med Hyg47:265-70; Mohan, B. et al. 2000 J Trop Pediatr 46:40-3; Wahid, S. F. etal. 2000 Southeast Asian J Trop Med Public Health 31:259-63). DEN virusantigen-positive hepatocytes are seen surrounding areas of necrosis inthe liver of fatal cases (Couvelard, A. et al. 1999 Hum Pathol30:1106-10; Huerre, M. R. et al. 2001 Virchows Arch 438:107-15), fromwhich dengue virus sequences were identified using RT-PCR (Rosen, L. etal. 1999 Am J Trop Med Hyg 61:720-4). Of potential importance to theetiology of severe dengue virus infection, three studies havedemonstrated that the mean levels of serum ALT and AST weresignificantly increased in patients with DHF/DSS compared to those withDF (Kalayanarooj, S. et al. 1997 J Infect Dis 176:313-21; Mohan, B. etal. 2000 J Trop Pediatr 46:40-3; Wahid, S. F. et al. 2000 SoutheastAsian J Trop Med Public Health 31:259-63). As expected, elevation ofserum liver enzymes has previously been observed in clinical trials ofDEN virus vaccine candidates (Example 8; Eckels, K. H. et al. 1984 Am JTrop Med Hyg 33:684-9; Edelman, R. et al. 1994 J Infect Dis 170:1448-55;Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-3188; Vaughn, D. W. et al.1996 Vaccine 14:329-36).

Based on the increasing disease burden associated with DEN virusinfection over the past several decades, a vaccine which confersprotection against the four dengue virus serotypes is needed, but noneis presently licensed. Because of the increased risk for severe DHF/DSSassociated with secondary infection with a heterologous DEN virusserotype (Burke, D. S. et al. 1988 Am J Trop Med Hyg 38:172-80;Halstead, S. B. et al. 1977 J Exp Med 146:218-29; Thein, S. et al. 1997Am J Trop Med Hyg 56:566-72), an effective vaccine must confersimultaneous protection against each of the four DEN virus serotypes.Several approaches are presently being pursued to develop a tetravalentvaccine against the dengue viruses (Bancroft, W. H. et al. 1984 J InfectDis 149:1005-10; Bhamarapravati, N. & Sutee, Y. 2000 Vaccine 18:44-7;Butrapet, S. et al. 2000 J Virol 74:3011-9; Guirakhoo, F. et al. 2000 JVirol 74:5477-85; Huang, C. Y. et al. 2000 J Virol 74:3020-8;Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-3188). One such approach,a live-attenuated DEN4 vaccine candidate, termed 2AΔ30, was bothattenuated and immunogenic in a cohort of 20 volunteers (Example 8). Therecombinant 2AΔ30 virus contains a 30 nt deletion in the 3′ UTR whichremoves nucleotides 10,478-10,507 and was found to produce a low orundetectable level of viremia in vaccinees at a dose of 10⁵ PFU/vaccine.An asymptomatic rash was reported in 50% of volunteers, and the onlylaboratory abnormality observed was an asymptomatic, transient rise inthe serum ALT level in 5 of the 20 vaccinees. All 2AΔ30 vaccineesdeveloped serum neutralizing antibodies against DEN4 virus (mean titer:1:580), and 2AΔ30 was not transmitted to mosquitoes that fedexperimentally on vaccinees (Troyer, J. M. et al. 2001 Am J Trop Med Hyg65:414-9). Because of the desirable properties conferred by the Δ30mutation, chimeric vaccine candidates are being constructed whichcontain the structural genes of DEN virus type 1, 2, and 3, in theattenuated DEN4 background bearing the genetically stable Δ30 mutation.Attenuating mutations outside of the structural genes are particularlyattractive for inclusion in antigenic chimeric vaccine candidatesbecause they will not affect the infectivity or immunogenicity conferredby the major mediator of humoral immunity to DEN viruses, the envelope(E) protein.

The presence of rash and elevated ALT levels suggests that the 2AΔ30vaccine candidate may be slightly under-attenuated in humans. Similarly,many previous attempts to develop live attenuated dengue virus vaccineshave yielded vaccine candidates that were either over- orunder-attenuated in humans, some of which also induced elevation ofserum ALT and AST levels (Bhamarapravati, N. & Yoksan, S. 1997 in:Dengue and Dengue Hemorrhagic Fever D. J. Gubler & G. Kuno eds. pp.367-377 CAB International, New York; Eckels, K. H. et al. 1984 Am J TropMed Hyg 33:684-9; Innis, B. L. et al. 1988 J Infect Dis 158:876-80;Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-3188; McKee, K. T., Jr. etal. 1987 Am J Trop Med Hyg 36:435-42). Therefore, we have developed amenu of point mutations conferring temperature-sensitive (ts),small-plaque (sp), and attenuation (att) phenotypes capable ofattenuating DEN4 viruses to a varying degree (Example 1). We havepreviously described 20 mutant viruses that exhibit a ts, but not sp,phenotype in Vero cells or HuH-7 liver cells and that show attenuatedreplication in mouse brain (Example 1). Addition of such mutations to2AΔ30 or to other dengue virus vaccine candidates is envisioned asyielding vaccine candidates that exhibit a more satisfactory balancebetween attenuation and immunogenicity.

In the present Example, we have extended our analysis of the panel of1,248 DEN4 virus clones previously generated by mutagenesis with5-fluorouracil (5-FU) (Example 1), by identifying a set of 22 sp mutantviruses, some of which also have a ts phenotype. Small plaque mutantviruses were sought since such viruses are often attenuated in humans(Bhamarapravati, N. & Yoksan, S. 1997 in: Dengue and Dengue HemorrhagicFever D. J. Gubler & G. Kuno eds. pp. 367-377 CAB International, NewYork; Butrapet, S. et al. 2000 J Virol 74:3011-9; Crowe, J. E. Jr. etal. 1994 Vaccine 12:783-790; Crowe, J. E. Jr. et al. 1994 Vaccine12:691-699; Eckels, K. H. et al. 1980 Infect Immun 27:175-80; Innis, B.L. et al. 1988 J Infect Dis 158:876-80; Murphy, B. R. & Chanock, R. M.2001 in: Fields Virology D. M. Knipe, et al. Eds. Vol. 1, pp. 435-468Lippincott Williams & Wilkins, Philadelphia; Takemoto, K. K. 1966 ProgMed Virol 8:314-48). Because natural infection with dengue viruses andvaccination with 2AΔ30 may be associated with liver toxicity in humans,we identified mutant viruses with restricted replication in human livercells. Accordingly, viruses were screened for plaque size andtemperature-sensitivity in the human hepatoma cell line, HuH-7, as wellas in Vero cells. Here we describe the ts phenotype, nucleotidesequence, and growth properties in suckling mice of 22 sp DEN4 mutantvirus clones.

Cells and Viruses.

WHO Vero cells (African green monkey kidney cells) and HuH-7 cells(human hepatoma cells) (Nakabayashi, H. et al. 1982 Cancer Res42:3858-63) were maintained as described in Example 1. DEN4 2A virus isa wild type virus derived from a cDNA clone of DEN4 strain 814669(Dominica, 1981) (Lai, C. J. et al. 1991 PNAS USA 88:5139-43; Mackow, E.et al. 1987 Virology 159:217-28). The nucleotide sequence of DEN4 2A,the parent of the 5-FU mutant viruses, was previously assigned GenBankaccession number AF375822 (Example 1). The DEN4 vaccine candidate,2AΔ30, (Example 8) contains a 30 nt deletion in the 3′ untranslatedregion (UTR) which removes nucleotides 10,478-10,507 (Men, R. et al.1996 J Virol 70:3930-7). The cDNA clones p4, a modified derivative ofthe DEN4 2A cDNA clone, and p4Δ30 were used to generate recombinant wildtype and attenuated viruses, rDEN4 and rDEN4Δ30, respectively (Example8). GenBank accession numbers were previously assigned as follows(virus: accession number): DEN4 strain 814669: AF326573; 2AΔ30:AF326826; rDEN4: AF326825; rDEN4Δ30: AF326827.

Generation and Biological Cloning of Mutant Viruses with a sp Phenotype.

The generation of 1,248 virus clones from a pool of5-fluorouracil-mutagenized DEN4 2A has been previously described(Example 1). Briefly, monolayers of Vero cells were infected with DEN42A at a multiplicity of infection (MOI) of 0.01 and overlaid with MEMsupplemented with 2% FBS and 1 mM 5-fluorouracil (5-FU) (Sigma, St.Louis, Mo.), which reduced replication of DEN4 2A 100-fold. Vero cellsin 96-well plates were inoculated with the 5-FU treated virussuspension, and virus clones were harvested from plates receivingterminally-diluted virus. A total of 1,248 virus clones were generatedfrom the cultures treated with 1 mM 5-FU. Two virus clones, 2A-1 and2A-13, were generated in the same manner from control cultures nottreated with 5-FU and served as parallel-passaged control viruses with awild type phenotype.

Evaluation of In Vitro Plaque Size and Temperature Sensitivity.

The 1,248 5-FU-mutagenized virus clones were screened for temperaturesensitivity by assessing virus replication at 35° C. (permissivetemperature) and 39° C. (restrictive temperature) in Vero and HuH-7cells. Cell monolayers in 96-well plates were inoculated with serialten-fold dilutions of virus and replicate plates were incubated at 35°C. and 39° C. for five days in temperature-controlled water baths. Virusreplication was determined by immunoperoxidase staining as previouslydescribed (Example 1). A collection of 193 5-FU virus clonesdemonstrated a 100-fold or greater reduction in titer at 39° C. ineither cell line, and these presumptive is viruses were furthercharacterized. The efficiency of plaque formation (EOP) at permissiveand restrictive temperatures and the plaque size of each of the 193virus clones were determined as follows. Serial ten-fold dilutions ofvirus suspension were inoculated onto confluent Vero cell and HuH-7 cellmonolayers in replicate 24-well plates. After incubation at 35° C. fortwo hours, monolayers were overlaid with 0.8% methylcellulose (EMScience, Gibbstown, N.J.) in L-15 medium (Quality Biologicals,Gaithersburg, Md.) supplemented with 2% FBS, gentamicin, andL-glutamine. After incubation of replicate plates for five days at 35,37, 38, or 39° C. in temperature-controlled water baths, plaques werevisualized by immunoperoxidase staining and counted as previouslydescribed. Plaque size of each of the 193 viruses was evaluated at thepermissive temperature (35° C.) and compared to that of DEN4 2A-13parallel-passaged control virus with a wild type plaque size. Mutantviruses incubated at the permissive temperature of 35° C. which had aplaque size ≦1 mm or ≦0.4 mm (approximately ≦50% the size of wild typeDEN4 2A-13) in Vero or HuH-7 cells, respectively, were designated ashaving a sp phenotype. The level of temperature sensitivity and plaquesize of each virus was confirmed in at least two separate experiments.Seventy-five viruses which were confirmed to have a putative ts and/orsp phenotype were biologically cloned an additional two times andphenotypes were re-assessed. Twenty-two of the 75 terminally dilutedviruses were found to have a sp phenotype. Sixteen of the 22 sp mutantviruses were also found to have a ts phenotype as defined by a 2.5 or3.5 log₁₀ PFU/ml reduction in virus titer in Vero or HuH-7 cells,respectively, at restrictive temperature compared to the permissivetemperature of 35° C. as previously described (Example 1). Twenty of the75 terminally-diluted viruses were found to have a ts phenotype withouta sp phenotype and were previously described (Example 1). The remainderof the 75 viruses did not meet either criteria for a ts or sp mutantvirus.

Evaluation of Sp Mutant Viruses for Restricted Replication in SucklingMice.

Animal experiments were carried out in accordance with the regulationsand guidelines of the National Institutes of Health, Bethesda, Md.Growth of DEN4 5-FU mutant viruses was determined in Swiss Webstersuckling mice (Taconic Farms, Germantown, N.Y.). Groups of sixseven-day-old mice were inoculated intracerebrally with 10⁴ PFU of virusin 30 μl Opti-MEM I (Invitrogen) and the brain of each mouse was removedfive days later and individually analyzed as previously described(Example 1). Clarified supernatants of 10% suspensions of mouse brainwere frozen at −70° C., and the virus titer was determined by plaqueassay in Vero cells.

Determination of the Complete Genomic Sequence of the Sp Mutant Viruses.

The nucleotide sequence of the 5-FU-mutagenized DEN4 viruses wasdetermined as described in Example 8. Briefly, genomic RNA was isolatedfrom virus clones and cDNA was prepared by reverse transcription andserved as template for the generation of overlapping PCR fragments. Apanel of primers was designed to sequence both strands of the PCRproduct from which consensus sequences were assembled and analyzed. Thenucleotide sequence of the 5′ and 3′ regions of the virus genome wasdetermined after circularization of the RNA genome as described inExample 8.

Identification of DEN45-Fluorouracil Mutant Viruses with a sp Phenotype.

The generation of a panel of 1,248 virus clones from a wild type DEN4 2Avirus suspension mutagenized by 5-FU has been described previously(Example 1). In the present study twenty-two mutant viruses with a spphenotype were identified. The plaque size of representative mutantviruses is illustrated in FIG. 3. The plaque size of DEN4 2A-13 virus (aparallel-passaged virus with a wild type phenotype derived from controlcultures not treated with 5-FU) was consistently smaller in HuH-7 cellsthan that observed in Vero cells (FIG. 3A). Mutant viruses #569 and#1189 (FIG. 3B) were sp in both Vero and HuH-7 cells. In contrast, 5-FUmutant virus clones #311 and #1083 (FIG. 3C) were sp in only HuH-7cells, suggesting a liver cell-specific defect in replication withinthis phenotypic group. As indicated in Table 7, five mutant viruses werefound to have a sp phenotype in both Vero and HuH-7 cells while 17viruses had a sp phenotype in only HuH-7 cells. Each 5-FU mutant virusclone was compared for a sp or ts phenotype with three control viruses,2A-13, wild type rDEN4, and rDEN4Δ30. The recombinant viruses, rDEN4 andrDEN4Δ30, each had a plaque size in Vero and HuH-7 cells similar to thatof DEN4 2A-13 indicating that the Δ30 mutation does not confer a spphenotype (Table 7).

Most of the sp 5-FU mutant viruses also had a ts phenotype in Veroand/or HuH-7 cells (Table 7) since mutant viruses were initiallyscreened for temperature sensitivity. Temperature-sensitivity wasdefined as a 2.5 or 3.5 log₁₀ PFU/ml reduction in virus titer in Vero orHuH-7 cells, respectively, at restrictive temperature compared to thepermissive temperature of 35° C. as previously defined (Example 1).Three mutant viruses (#574, #1269 and #1189) were sp and ts in both Veroand HuH-7 cells, while nine mutant viruses (#506-326 in Table 7) werefound to be ts in both cell types but sp only in HuH-7 cells. Fourviruses (#1104, 952, 738, and 1083) were found to have a wild typephenotype in Vero cells but were both sp and ts in HuH-7 cells. Thesefour mutant viruses each had a 6,000- to 600,000-fold reduction in virustiter at 39° C. in HuH-7 cells with only a 6- to 40-fold reduction at39° C. in Vero cells. Finally, sp mutant viruses were identified whichdid not have a ts phenotype in either cell line; two of these viruses(#569 and #761) were sp in both Vero and HuH-7 cells and four viruses(#1096-1012) were sp in only HuH-7 cells (Table 7). As describedpreviously, the Δ30 mutation did not confer temperature-sensitivity ineither cell line (Example 1).

The sp 5-FU Mutant Viruses have Restricted Replication in Suckling MouseBrain.

The 22 sp DEN4 5-FU mutant viruses were evaluated for their ability toreplicate in the brain of one-week-old suckling mice. As a marker for invivo attenuation, their level of replication was compared with that ofthe parallel-passaged control virus with a wild type phenotype, 2A-13(Table 7). Nineteen of 22 sp mutant viruses had a greater than 100-foldreduction in virus replication in the brain of suckling mice compared to2A-13 and nine viruses had a reduction of greater than 10,000-fold.

The five mutant viruses which were sp in both Vero and HuH-7 cells were5,000-fold to 100,000-fold restricted in replication compared to 2A-13.Two of these mutant viruses, #569 and #761, were not ts in either cellline but had a reduction in virus titer of greater than 10,000-fold inmouse brain, indicating that the sp phenotype in both Vero and HuH-7cells is an important surrogate marker for attenuated replication insuckling mouse brain. 5-FU mutant viruses which were sp in only HuH-7cells had a more variable range of replication in mouse brain. Threeviruses had a mean reduction in virus titer of less than 10-fold whencompared to 2A-13 virus. However, 8 of 13 viruses which were ts in Veroand/or HuH-7 cells but sp in only HuH-7 cells had a greater than5,000-fold reduction in virus replication. The results of the in vivoreplication analysis of the previously described 20 ts 5-FU mutantviruses (Example 1) and the 22 sp mutant viruses are summarized in Table8. Mutant viruses with both a sp and ts phenotype were found to have asignificantly greater level of attenuation in the brain of suckling micewhen compared to viruses with only a ts phenotype.

Sequence Analysis of the sp 5-FU Mutant Viruses.

To initiate an analysis of the genetic basis of the ts, sp, or attphenotype of the 22 sp mutant viruses, the complete nucleotide sequenceof each virus genome was determined and is summarized in Table 9 (sp inVero and HuH-7 cells) and Table 10 (sp in only HuH-7 cells). Allidentified mutations were nucleotide substitutions, as deletions orinsertions were not observed. Point mutations were distributedthroughout the genome, including the 3′ UTR as well as in all codingregions. Because all 5-FU mutant viruses were found to have at least twomutations (two to six), the observed phenotypes cannot be directlyattributed to a specific mutation. The majority of sp viruses alsocontained translationally silent point mutations (none to four) in thestructural or non-structural coding regions. However, these silentmutations are not expected to contribute to the observed phenotypes. Sixof the 22 sp mutant viruses (Tables 9 and 10) were found to havemutations in only the NS genes and/or the 3′ UTR, indicating that the spphenotype can be conferred by mutations outside of the structural genes.

Presence of Identical Mutations in Multiple 5-FU Mutant Viruses.

Analysis of the complete nucleotide sequence data for the 5-FU mutantviruses identified several repeated mutations which were present in twoor more viruses. Such mutations were also identified previously duringour analysis of twenty 5-FU mutant viruses with a ts but not spphenotype (Example 1). Because these mutations occurred in virusestogether with additional mutations, the contribution of the repeatedmutations to the observed sp, ts, and att phenotypes remains empirical.Table 11 lists the repeated mutations found among the 20 ts (not sp)mutant viruses described previously (Example 1) and the 22 sp mutantviruses described here. Repeated mutations were identified in thefollowing genes: two in E, two in NS3, five in NS4B, one in NS5, and twoin the 3′ UTR. Interestingly, within a thirty nucleotide region of NS4B(nt 7153-7182), there were five different nucleotide substitutions whichwere found in sixteen viruses. Also at nt 7,546 in NS4B, an amino acidsubstitution (Ala→Val) was found in 10 different 5-FU mutant viruses.The significance of these repeated mutations in NS4B as well as in otherDEN4 genomic regions remains empirical, but a reasonable explanation forthis phenomenon is that these mutations are involved in adaptation ofDEN4 virus for efficient growth in Vero cells, as further discussed inExample 6.

Discussion.

As part of a molecular genetic vaccine strategy, we have developedattenuating mutations that are envisioned as being useful in thedevelopment of a live attenuated tetravalent dengue virus vaccine.Specifically, mutations which restrict replication of the vaccine virusin human liver cells were generated since there was some residualvirulence of the rDEN4Δ30 vaccine candidate for the liver of humans.Mutant viruses with a sp phenotype were sought in both Vero cells andHuH-7 human liver cells, in order to identify host-range mutant virusesthat were specifically restricted in replication in HuH-7 cells (sp inHuH-7 but not in Vero). Such mutations are envisioned as being useful inlimiting replication of a candidate vaccine in the liver of vaccineswhile preserving both efficient replication in Vero cells andimmunogenicity in vivo.

Several observations from the present study indicate that sp mutationsconfer an att phenotype in vivo. This is not surprising sinceattenuation in suckling mouse brain has been reported for live DEN virusvaccine candidates possessing sp phenotypes, including the DEN2 PDK-53and DEN2 PR-159/S-1 vaccine strains (Bhamarapravati, N. & Yoksan, S.1997 in: Dengue and Dengue Hemorrhagic Fever D. J. Gubler & G. Kuno eds.pp. 367-377 CAB International, New York; Butrapet, S. et al. 2000 JVirol 74:3011-9; Eckels, K. H. et al. 1980 Infect Immun 27:175-80;Innis, B. L. et al. 1988 J Infect Dis 158:876-80). Each of 22 DEN4 5-FUmutant viruses with a sp phenotype (some of which were also ts) ineither Vero or HuH-7 cells manifested restricted replication in thebrains of mice. Six 5-FU mutant viruses with a sp phenotype in theabsence of a ts phenotype were more attenuated in the brains of sucklingmice than mutant viruses with solely a ts phenotype (Example 1),indicating that the sp phenotype specifies a greater level ofattenuation for mouse brain than does the ts phenotype. Mutant viruseswith both a ts and sp phenotype had an even greater reduction inreplication, further indicating that the attenuation conferred by the tsand sp phenotypes can be additive. Importantly, seventeen of the 22 spmutant viruses were host-range sp mutant viruses, being sp only in HuH-7cells. Since such mutations are envisioned as being useful inrestricting the replication of a DEN4 virus in human liver cells, weused nucleotide sequence analysis to determine the genetic basis of thesp phenotype.

Analysis of the complete genomic sequence of the 22 sp DEN4 virusesrevealed substitutions in the 3′ UTR as well as coding mutations in allgenes except NS4A. It was first noted that several specific mutationswere present in two or more of the 22 sp DEN4 mutant viruses and thatmany of these same mutations were also previously identified among theset of 20 ts DEN4 mutant viruses (Example 1). Since flaviviruses canrapidly accumulate mutations during passage in tissue culture (Dunster,L. M. et al. 1999 Virology 261:309-18; Mandl, C. W. et al. 2001 J Virol75:5627-37), many of these over-represented mutations, previouslyreferred to as putative Vero cell adaptation mutations (Example 1),likely promote efficient replication in Vero cells and were selectedunintentionally during the biological cloning of the mutant viruses. Theeffect of these mutations on DEN virus replication in Vero cells, theproposed substrate for vaccine manufacture, is discussed in Example 6.

The sp mutations identified among the 5-FU mutant viruses are envisionedas being useful in several different approaches for the development ofDEN virus vaccine strains. As described above for the generation ofantigenic chimeric viruses, one or more sp attenuating mutations areenvisioned as being added to the attenuated DEN4Δ30 genetic backgroundto supplement the att phenotype of the Δ30 mutation. A second approachis to introduce a sp attenuating mutation, with or without Δ30, intoinfectious cDNA clones of the other three DEN serotypes. The ability totransfer mutations among genetically-related viruses and maintainsimilar att phenotypes has been previously demonstrated (Skiadopoulos,M. H. et al. 1999 Virology 260:125-35). These distinct strategies areenvisioned as being useful as separate or complementary approaches tothe construction of a tetravalent DEN virus vaccine, underlining theimportance of the identification of a large panel of att mutationswithin the DEN viruses.

Example 3 Recombinant DEN4 Viruses Containing Mutations Identified in5-FU Mutant Viruses Show Restricted Replication in Suckling Mouse Brainand in SCID Mice Transplanted with Human Liver Cells

Data was presented in Examples 1 and 2 that summarizes the generation,characterization and sequence analysis of 42 attenuated mutant DEN4viruses. For three of the mutant viruses (#239, 489, and 773) with asingle missense mutation at nt position 4995 in NS3, it was clear thatthe identified mutation specified the ts and att phenotypes. Thisconclusion was confirmed in Example 1 by tissue culture and in vivocharacterization of rDEN4-4995, a recombinant virus into which the 4995mutation had been introduced by site-directed mutagenesis. In thisanalysis, rDEN4-4995 exhibited the same level of temperature sensitivityand attenuation as 5-FU mutant viruses #239, 489, and 773. Theindividual mutation(s) in the remaining 5-FU mutant viruses that specifythe observed phenotypes remains to be identified, since most of theseviruses possess more than one nucleotide substitution. We have conductedan analysis to identify the mutations in a subset of the other 39 mutantviruses that specify the ts, sp, and att phenotypes by introduction ofeach mutation into the wt DEN4 cDNA (p4) and evaluation of thephenotypes of the resulting recombinant DEN4 viruses bearing theindividual mutations. Previous studies of a DEN2 virus vaccine candidate(Butrapet, S. et al. 2000 J Virol 74:3011-9) as well as other virusvaccines (Whitehead, S. S. et al. 1999 J Virol 73:871-7) havedemonstrated the utility of this approach for the identification of thegenetic basis of attenuation.

As described in Examples 1 and 2, 19 5-FU mutant viruses were identifiedwhich were found to contain coding mutations in only the NS genes and/ornucleotide substitutions in the 5′ or 3′ UTR which would facilitate thegeneration of antigenic chimeric viruses. In the present example, thegenetic basis of the observed sp, ts, and mouse brain att phenotypes wasidentified for these 19 viruses using reverse genetics to generaterecombinant DEN4 (rDEN4) viruses containing individual mutationsidentified in the panel of DEN4 mutant viruses. In addition, the 19 5-FUmutant viruses were evaluated for replication in a novel small animalmodel for DEN4 virus replication, SCID mice transplanted with HuH-7cells (SCID-HuH-7), and the genetic basis of the att viruses wasidentified using mutant rDEN4 viruses. Also presented are findingsdescribing the generation and characterization of a recombinant viruscontaining two of the identified attenuating mutations as well ascombination of select 5-FU mutations with the Δ30 mutation.

Generation of rDEN4 Viruses Containing 5-FU Mutations.

The methods used for the generation of rDEN4 viruses are outlined inFIG. 4 and are similar to those described in Example 1. Briefly, the p4cDNA was digested with the appropriate restriction enzymes and theresulting fragments were subcloned into a modified pUC119 vector. ForKunkel mutagenesis, single-stranded DNA preparations of the pUC-NSvectors were made, and primers were designed to individually introducemutations that were present in the 5-FU mutant viruses. The sequences ofthe 41 mutagenic oligonucleotides used to generate the single-mutationrecombinant viruses are presented in Table 12. Primers were designed toco-introduce or co-ablate a translationally-silent restriction enzymesite in the cDNA, which greatly facilitates the screening andidentification of cDNA clones possessing the mutant sequence. Fragmentscontaining the introduced mutations were cloned back into p4, andnucleotide sequence analysis confirmed the presence of the nucleotidechanges. A total of 33 rDEN4 viruses was generated which contained eachof the individual mutations present in the 19 5-FU mutant virusescontaining only coding mutations in the NS genes and/or nucleotidesubstitutions in the 5′ or 3′ UTR. An additional 8 rDEN4 viruses weregenerated from mutations identified in the remaining panel of 42 5-FUmutant viruses.

A cDNA clone was also generated which combined the mutations identifiedat nt position 4995 in NS3 and 7849 in NS5. The 7849 mutation wasintroduced into the p4-4995 cDNA clone by replacing the XmaI-PstIfragment with that derived from the p4-7849 cDNA clone. The presence ofboth mutations was confirmed by sequence analysis. The Δ30 mutation wasintroduced into the 3′ UTR of the individual mutant cDNA clones byreplacing the MluI-KpnI fragment with that derived from the p4Δ30 cDNAclone, and the presence of the deletion was confirmed by sequenceanalysis.

Recombinant viruses were recovered by transfection of Vero or C6/36cells with RNA transcripts derived from the mutant cDNA clones asdescribed in Example 1. Recovered viruses were terminally diluted twiceand working stocks of viruses were prepared in Vero cells. Each of themutant cDNA clones was recovered after transfection as expected sincethe 5-FU mutant viruses containing these mutations were viable.

Characterization of ts and att Phenotypes of the rDEN4 VirusesContaining Introduced Mutations.

Of the 19 5-FU mutant viruses with mutations in only NS genes and/or the5′ or 3′ UTR, six had an sp phenotype (Table 13), ten had a ts phenotypein Vero and HuH-7 cells (Table 14), and three had a ts phenotype in onlyHuH-7 cells (Table 15). For the six sp 5-FU mutant viruses, #738, 922,1081, 1083, 1136, and 1189, seventeen mutations identified by sequenceanalysis resulted in a coding change or a nucleotide change in the UTRand each was engineered into an individual DEN4 cDNA clone. Viruscontaining each defined mutation was successfully recovered andpropagated and was tested for efficiency of plaque formation in Vero andHuH-7 cells at various temperatures, plaque size phenotype, and growthproperties in suckling mice using methods previously described inExamples 1 and 2.

Table 13 lists the phenotypes of the six sp 5-FU mutant parent virusesand those of the 17 rDEN4 viruses encoding single mutations present inthe parent virus. For example, 5-FU mutant #1189 (parent), which was tsand sp in both cell lines and had an almost 10,000-fold reduction inreplication in suckling mouse brain, contained 4 coding mutations at ntposition 3303 in NS1, 4812 and 5097 in NS3, and 7182 in NS4B. Analysisof the four rDEN4 viruses containing each of these mutations indicatedthat rDEN4-5097 had a ts, sp, and att phenotype while rDEN4-3303,rDEN4-4812, and rDEN4-7182 had no discernible phenotypes, indicatingthat the mutation at nt 5097 was responsible for the phenotype observedin the 5-FU parent, #1189. Thus, analysis of the relative contributionsof the four mutations present in the 5-FU mutant #1189 to itsattenuation phenotype provides the framework for a similar analysis ofthe remaining 5-FU mutant viruses. This analysis specificallydemonstrates the methods used to identify mutations contributing to theobserved phenotype. The ts, sp, and att phenotypes of 5-FU parentviruses #738, 922, 1081, and 1083, were similarly attributed to singlemutations 3540, 4306, 2650, and 10634, respectively. However, twoseparate mutations (3771 and 4891) contributed to the phenotypes of 5-FUmutant virus #1136.

Table 14 lists the genetic basis of the ts and mouse brain attenuationfor the ten 5-FU mutant viruses with ts phenotypes in both Vero andHuH-7 cells. As described in Example 1, the 4995 mutation which is theonly mutation present in three 5-FU mutant viruses, #239, #489, and#773, was found to confer a ts and att phenotype, confirming the geneticbasis for the phenotypes exhibited by these viruses. In three separateexperiments, the rDEN4-4995 virus was found to have an approximately1,000-fold decrease in replication in the brains of suckling mice whencompared to that of wild-type virus (Table 6 and 14). The 4995 mutationis also present in 5-FU mutant viruses #473, #759, and #816, each ofwhich has additional mutations. The ts and att phenotypes observed inthese viruses can be attributed to the 4995 mutation since theadditional mutations did not show discernible phenotypes. Interestingly,5-FU mutant virus #938 has the 4995 mutation and an additional mutationat nt 3442 in NS1 with both mutations independently conferringrestricted replication in mouse brain. The remaining three 5-FU parentviruses in Table 14, #173, #509, and #1033, were found to each contain asingle mutation responsible for the att phenotype: 7849, 8092, and 4907,respectively.

Three 5-FU mutant viruses, #686, #992, and #1175 with HuH-7cell-specific is phenotypes are listed in Table 15. Mutations in NS3(5695) and NS5 (10186) were found to confer the phenotypes observed forparent virus #992 and #1175. Interestingly, two mutations in NS2A, 3575and 4062, were found to result in a synergistic increase in the level ofattenuation. Both individual mutations had an approximately 100-folddecrease in virus replication in the brain while the parent virus withboth mutations had an almost 10,000-fold reduction. Table 16 lists twoadditional mutations with an att phenotype, 4896 and 6259 in NS3.

Replication of DEN4 Viruses in SCID Mice Transplanted with HuH-7 Cells.

Since DEN viruses replicate poorly in the liver of mice andcorresponding studies are impractical to conduct in non-human primates,an animal model that evaluates the in vivo level of replication of DENvirus in liver cells was developed based on a recent report examiningthe replication of DEN virus in SCID mice transplanted with a continuouscell line of human liver tumor cells (An, J. et al. 1999 Virology263:70-7). SCID mice transplanted with human continuous cell lines,primary cells, or organized tissues have similarly been used to studythe replication of other viruses which lack a suitable small animalmodel (Mosier, D. E. 2000 Virology 271:215-9). In our study, SCID micewere transplanted with HuH-7 cells since DEN4 virus replicatedefficiently in these cells in tissue culture and since these were thecells used to define the host-range phenotype. These studies areenvisioned as addressing the utility of examining DEN virus infection inSCID mouse-xenograft models for vaccine development (An, J. et al. 1999Virology 263:70-7; Lin, Y. L. et al. 1998 J Virol 72:9729-37).

To further examine the in vivo growth properties of the 19 5-FU mutantDEN4 viruses with mutations in only the NS genes and/or the 3′ UTR andselected corresponding rDEN4 mutant viruses, replication was assessed inSCID mice transplanted with HuH-7 cells (SCID-HuH-7). For analysis ofDEN4 virus replication in SCID-HuH-7 mice, four to six week-old SCIDmice (Tac:Icr:Ha(ICR)-Prkdc^(scid)) (Taconic Farms) were injectedintraperitoneally with 10⁷ HuH-7 cells suspended in 200 μlphosphate-buffered saline (PBS). In preparation for transplantation,HuH-7 cells were propagated in cell culture as described above andharvested by trypsinization at approximately 80% confluence. Cells werewashed twice in PBS, counted, resuspended in an appropriate volume ofPBS, and injected into the peritoneum of mice. Tumors were detected inthe peritoneum five to six weeks after transplantation, and only micewith apparent tumors were used for inoculation. Mice were infected bydirect inoculation into the tumor with 10⁴ PFU of virus in 50 μlOpti-MEM I. Mice were monitored daily for seven days and serum for virustitration was obtained by tail-nicking on day 6 and 7. Approximately 400μl blood was collected in a serum separator tube (Sarstedt, Germany),centrifuged, and serum was aliquoted and stored at −70° C. The virustiter was determined by plaque assay in Vero cells. Seven days afterinfection, most mice developed morbidity and all mice were sacrificed.Tumors were excised and weighed to confirm uniformity of theexperimental groups.

Preliminary experiments indicated that SCID-HuH-7 mice inoculated withDEN4 2A-13 directly into the tumor developed viremia with maximum levels(up to 8.0 log₁₀ PFU/ml serum) achieved on day 5 (Table 17). Virus couldalso be detected in brain, liver, and tumor homogenates.

The level of viremia in SCID-HuH-7 mice infected with parental 5-FU orrDEN4 mutant viruses was compared with that of the parallel-passagedcontrol virus, 2A-13, or rDEN4, respectively. Results of 4 separateexperiments indicated that the vaccine candidate, rDEN4Δ30, had analmost 10-fold reduction in virus replication compared to wild typerDEN4 (Table 13) which reflects the apparent attenuation of the rDEN4Δ30vaccine candidate in humans (Example 8). Results in Tables 13 to 15indicate that three 5-FU mutant viruses had a greater than 100-foldreduction in viremia in the SCID-HuH-7 mice compared to wild type 2A-13virus: #1081, #1083, and #1189. The common phenotype among these viruseswas asp phenotype in HuH-7 cells. Analysis of the genetic basis of theatt phenotype in these parent 5-FU mutant viruses identified threeindividual mutations in NS1, NS3, and the 3′ UTR which conferred atleast a 100-fold reduction in viremia. Specifically, rDEN4-2650 (NS1),rDEN4-5097 (NS3), and rDEN4-10634 (3′ UTR) manifested a 2.2, 3.6, and4.3 log₁₀ PFU/ml reduction in peak titer of viremia compared to rDEN4,respectively. These mutations also conferred the att phenotype insuckling mouse brain. 5-FU mutant virus #738 and #509 had a reduction inviremia in the SCID-HuH-7 mice compared to wild type 2A-13 of 1.9 and1.5 log₁₀ PFU/ml, respectively, and the genetic basis for thesephenotypes is envisioned as being assessed on an empirical basis.

This analysis of the genetic basis of the phenotypes specified by themutations in the 5-FU mutant viruses that manifested restrictedreplication in SCID-HuH-7 mice indicated that (1) three separatemutations conferred the att phenotype; (2) these mutations were locatedin two proteins, NS1 and NS3, and in the 3′ UTR; (3) these threemutations were fully responsible for each of the cell culture (ts or sp)and in vivo (attenuation in mouse brain and SCID-HuH-7 mice) phenotypesof the parent viruses; and (4) two of the three mutations specify thehost-range sp phenotype (sp on HuH-7 only) and therefore are envisionedas being useful in a vaccine virus. Although the relevance of suchSCID-transplant models to virus replication and disease in humans isunknown, the identification of three novel mutations which restrict DEN4virus replication in SCID-HuH-7 mice is envisioned as facilitating anexamination of the correlation between the att phenotype in SCID-HuH-7mice with that in rhesus monkeys or humans. Such mutations, specificallythe host-range sp mutations, are envisioned as being useful inconjunction with the Δ30 or other mutation to decrease the residualvirulence of rDEN4Δ30 or other dengue virus for the human liver, andstudies are envisioned as being conducted to construct such rDEN4viruses and evaluate them in monkeys and humans (Example 8).

Combination of Two 5-FU Mutations Results in an Additive ts Phenotype.

The ability to combine individual mutations in rDEN4 virus as a means tomodulate the phenotype of the resulting double mutant virus is a majoradvantage of using recombinant cDNA technology to generate or modifydengue virus vaccine candidates. Addition of multiple ts and attmutations to recombinant vaccine viruses is envisioned as improving thephenotypic stability of the double recombinant due to the decreasedpossibility of co-reversion of the two mutations to wild-type virulence(Crowe, J. E. Jr. et al. 1994a Vaccine 12:783-790; Skiadopoulos, M. H.et al. 1998 J Virol 72:1762-8; Subbarao, E. K. et al. 1995 J Virol69:5969-5977; Whitehead, S. S. et al. 1999 J Virol 73:871-7). Themutations identified at nt position 4995 in NS3 and 7849 in NS5 werecombined in a single p4 cDNA clone and a recombinant virus, designatedrDEN4-4995-7849, was recovered and evaluated for its ts and attphenotypes (Table 18). rDEN4-4995-7849 was more ts than eitherrecombinant virus containing the individual mutations (Table 18),indicating the additive effect of the two ts mutations. TherDEN4-4995-7849 virus had a greater than 10,000-fold reduction inreplication in the brains of suckling mice. The reduction in replicationof the double mutant virus was only slightly increased over that ofrDEN4-7849, however, a difference in the level of replication betweenrDEN4-4995-7849 and rDEN4-7849 would be difficult to detect since thelevel of replication of both viruses was close to the lower limit ofdetection (2.0 log₁₀ PFU/g brain).

Combination of Selected 5-FU Mutations with the Δ30 Mutation ConfersIncreased Attenuation of rDEN4Δ30 for the Brains of Suckling Mice.

To define the effect of adding individual mutations to the attenuatedrDEN4Δ30 background, five combinations have been constructed:rDEN4Δ30-2650, rDEN4Δ30-4995, rDEN4Δ30-5097, rDEN4Δ30-8092, andrDEN4Δ30-10634. Addition of such missense mutations with various ts, sp,and att phenotypes is envisioned as serving to decrease thereactogenicity of rDEN4Δ30 while maintaining sufficient immunogenicity.

The Δ30 mutation was introduced into the 3′ UTR of the individual mutantcDNA clones by replacing the MluI-KpnI fragment with that derived fromthe p4Δ30 cDNA clone, and the presence of the deletion was confirmed bysequence analysis. Recombinant viruses were recovered by transfection inC6/36 cells for each rDEN4 virus. However, upon terminal dilution andpassage, the rDEN4Δ30-5097 virus was found to not grow to a sufficienttiter in Vero cells and was not pursued further. This is an example of acDNA in which the 5-FU mutation and the 030 mutation are not compatiblefor efficient replication in cell culture. To begin the process ofevaluating the in vivo phenotypes of the other four viruses whichreplicated efficiently in cell culture, rDEN4 viruses containing theindividual mutations and the corresponding rDEN4Δ30 combinations weretested together for levels of replication in suckling mouse brain. Theresults in Table 19 indicate that addition of each of the mutationsconfers an increased level of attenuation in growth upon the rDEN4Δ30virus, similar to the level conferred by the individual 5-FU mutation.No synergistic effect in attenuation was observed between the missensemutations and Δ30. These results indicate that the missense mutations atnucleotides 2650, 4995, 8092, and 10634 are compatible with Δ30 forgrowth in cell culture and in vivo and can further attenuate therDEN4Δ30 virus in mouse brain. Further studies in SCID-HuH-7 mice,rhesus monkeys, and humans are envisioned as establishing the effect ofthe combination of individual mutations and 030 upon attenuation andimmunogenicity (Example 8).

By identifying the specific mutations in the 5-FU mutant viruses whichconfer the observed phenotypes, a menu of defined ts, sp, and attmutations is envisioned as being assembled (see Example 7). Numerouscombinations of two or more of these mutations are envisioned as beingselected with or without the Δ30 mutation. Such mutations and theircombinations are envisioned as being useful for the construction ofrecombinant viruses with various levels of in vivo attenuation, thusfacilitating the generation of candidate vaccines with acceptable levelsof attenuation, immunogenicity, and genetic stability.

Example 4 Generation of DEN4 Mutant Viruses with Temperature-Sensitiveand Mouse Attenuation Phenotypes Through Charge-Cluster-to-AlanineMutagenesis

The previous Examples described the creation of a panel of DEN4 mutantviruses with ts, sp, and att phenotypes obtained through 5-FUmutagenesis. As indicated in these Examples, the attenuating mutationsidentified in the 5-FU mutant viruses are envisioned as having severaluses including (1) fine tuning the level of attenuation of existingdengue virus vaccine candidates and (2) generation of new vaccinecandidates by combination of two or more of these attenuating mutations.In the current example, we created a second panel of mutant virusesthrough charge-cluster-to-alanine mutagenesis of the NS5 gene of DEN4and examined the resulting mutant viruses for the ts, sp, and attphenotypes as described in Examples 1 and 2. Thecharge-cluster-to-alanine mutant viruses recovered demonstrated a rangeof phenotypes including ts in Vero cells alone, ts in HuH-7 cells alone,ts in both cell types, att in suckling mouse brains, and att inSCID-HuH-7 mice.

The usefulness of mutant viruses expressing these phenotypes has alreadybeen described, however charge-cluster-to-alanine mutant viruses possesssome additional desirable characteristics. First, the relevant mutationsare envisioned as being designed for use in the genes encoding thenon-structural proteins of DEN4, and therefore are envisioned as beinguseful to attenuate DEN1, DEN2, and DEN3 antigenic chimeric recombinantspossessing a DEN4 vector background. Second, the phenotype is usuallyspecified by three or more nucleotide changes, rendering the likelihoodof reversion of the mutant sequence to that of the wild type sequenceless than for a single point mutation, such as mutations identified inthe panel of 5-FU mutant viruses. Finally, charge-cluster-to-alanineattenuating mutations are envisioned as being easily combinable amongthemselves or with other attenuating mutations to modify the attenuationphenotype of DEN4 vaccine candidates or of DEN1, DEN2, and DEN3antigenic chimeric recombinant viruses possessing a DEN4 vectorbackground.

Charge-Cluster-to-Alanine-Mutagenesis.

The cDNA p4, from which recombinant wild type and mutant viruses weregenerated, has been described in Examples 1, 2, and 3 and in FIG. 4.Charge-cluster-to-alanine mutagenesis (Muylaert, I. R. et al. 1997 JVirol 71:291-8), in which pairs of charged amino acids are replaced withalanine residues, was used to individually mutagenize the codingsequence for 80 pairs of contiguous charged amino acids in the DEN4 NS5gene. Subclones suitable for mutagenesis were derived from the fulllength DEN4 plasmid (p4) by digestion with XmaI/PstI (pNS5A), PstI/SacII(pNS5B) or SacII/MluI (pNS5C) at the nucleotide positions indicated inFIG. 4. These fragments were then subcloned and Kunkel mutagenesis wasconducted as described in Examples 1 and 3. To create each mutation,oligonucleotides were designed to change the sequence of individualpairs of codons to GCAGCX (SEQ ID NO: 69), thereby replacing them withtwo alanine codons (GCX) and also creating a BbvI restriction site(GCAGC) (SEQ ID NO: 70). The BbvI site was added to facilitate screeningof cDNAs and recombinant viruses for the presence of the mutantsequence. Restriction enzyme fragments bearing the alanine mutationswere cloned back into the full-length p4 plasmid as described inExamples 1 and 3.

Initial evaluation of the phenotype of the 32 charge-cluster-to-alaninemutant viruses revealed a range in restriction of replication insuckling mouse brain and SCID-HuH-7 mice. To determine whetherattenuation could be enhanced by combining mutations, double mutantviruses carrying two pairs of charge-cluster-to-alanine mutations werecreated by swapping appropriate fragments carrying one pair of mutationsinto a previously-mutagenized p4 cDNA carrying a second pair ofmutations in a different fragment using conventional cloning techniques.

Transcription and Transfection.

5′-capped transcripts were synthesized in vitro from mutagenized cDNAtemplates using AmpliCap SP6 RNA polymerase (Epicentre, Madison, Wis.).Transfection mixtures, consisting of 1 μg of transcript in 60 μl ofHEPES/saline plus 12 μl of dioleoyl trimethylammonium propane (DOTAP)(Roche Diagnostics Corp., Indianapolis, Ind.), were added, along with 1ml Virus production-serum free medium (VP-SFM) to subconfluentmonolayers of Vero cells in 6-well plates. Transfected monolayers wereincubated at 35° C. for approximately 18 hr, cell culture medium wasremoved and replaced with 2 ml VP-SFM, and cell monolayers wereincubated at 35° C. After 5 to 6 days, cell culture medium wascollected, and the presence of virus was determined by titration in Verocells followed by immunoperoxidase staining as previously described.Recovered virus was amplified by an additional passage in Vero cells,and virus suspensions were combined with SPG(sucrose-phosphate-glutamate) stabilizer (final concentration: 218 mMsucrose, 6 mM L-glutamic acid, 3.8 mM potassium phosphate, monobasic,and 7.2 mM potassium phosphate, dibasic, pH 7.2), aliquoted, frozen ondry ice, and stored at −70° C.

cDNA constructs not yielding virus after transfection of Vero cells wereused to transfect C6/36 cells as follows. Transfection mixtures, asdescribed above, were added, along with 1 ml of MEM containing 10% fetalbovine serum (FBS), 2 mM L-glutamine, 2 mM non-essential amino acids,and 0.05 mg/ml gentamicin, to monolayers of C6/36 cells. Transfectedcell monolayers were incubated at 32° C. for 18 hr, cell culture mediumwas removed and replaced with 2 ml fresh medium, and cell monolayerswere incubated at 32° C. After 5 to 6 days, cell culture media were thenused to infect Vero cells and incubated for 5-6 days, at which time cellculture media were collected, frozen and titered as described above.

Recovered viruses were biologically cloned by two rounds of terminaldilution in Vero cells followed by an additional amplification in Verocells. Briefly, virus was initially diluted to a concentration ofapproximately 20 PFU/ml in VP-SFM and then subjected to a series oftwo-fold dilutions across a 96-well plate. Virus dilutions were used toinfect Vero cell monolayers in a 96-well plate and incubated for 5 to 6days at 35° C. Following incubation, cell culture media were removed andtemporarily stored at 4° C., and the virus-positive cell monolayers wereidentified by immunoperoxidase staining. Terminal dilution was achievedwhen ≦25% of cell monolayers were positive for virus. Cell culturemedium from a positive monolayer at the terminal dilution was subjectedto an additional round of terminal dilution. Following the secondterminal dilution, virus was amplified in Vero cells (75 cm² flask),collected and frozen as previously described.

Assays for Temperature-Sensitivity and Mouse Attenuation.

Assay of the level of temperature sensitivity of thecharge-cluster-to-alanine mutant viruses in Vero and HuH-7 cells andtheir level of replication in the brain of suckling mice were conductedas described in Example 1 and assay of the level of replication inSCID-HuH-7 mice was conducted as described in Example 3.

Charge-Cluster-to-Alanine Mutant Viruses are Viable and ShowTemperature-Sensitive and Mouse Attenuation Phenotypes.

Of 80 full-length DEN4 cDNA constructs containing a single pair ofcharge-to-alanine mutations, virus was recovered from 32 in either Veroor C6/36 cells (FIG. 5). The level of temperature sensitivity of wtrDEN4, rDEN4Δ30, and the 32 mutant viruses is summarized in Table 20.One mutant virus (645-646) was ts in Vero but not HuH-7 cells and 7mutant viruses were ts in HuH-7 but not Vero cells. Such mutants whosetemperature sensitivity is host-cell dependent are referred to astemperature-sensitive, host-range (tshr) mutants. Thirteen mutantviruses were ts in both cell types, and 11 mutant viruses were not ts oneither cell type. Thus a total of 21 mutant viruses were ts with 8mutant viruses exhibiting an tshr specificity. None of the mutantviruses showed a small plaque phenotype at permissive temperature.Mutant viruses showed a wide range (0 to 10,000-fold) of restrictedreplication in suckling mouse brain (Table 20). Fourteen mutant viruseswere attenuated in suckling mouse brain, arbitrarily defined as a ≧1.5log₁₀-unit reduction in virus titer. There was no correlation betweenattenuation in mouse brain and temperature sensitivity in either Verocells (Kendall Rank correlation: P=0.77) or HuH-7 cells (Kendall Rankcorrelation: P=0.06).

Thirteen mutant viruses that either showed an att phenotype in sucklingmouse brain or whose unmutated charged amino acid pair was highlyconserved among the four DEN serotypes (see Example 7) were assayed foratt in SCID-HuH-7 mice (Table 21). Three of these mutant virusesshowed >100-fold decrease in replication relative to wild type DEN4.Overall, mean log reduction from wild type in suckling mice did not showsignificant correlation with mean log reduction in SCID-HuH-7 mice(Spearman rank correlation, N=13, P=0.06). However, mutant virus 200-201was unusual in that it showed a high level of restriction in SCID-HuH-7mice but little restriction in suckling mouse brain. When virus 200-201was removed from the analysis, restriction of replication in sucklingand SCID-HuH-7 mice showed a significant correlation (Spearman rankcorrelation, N=12, P=0.02).

Combining Charge-Cluster-to-Alanine Mutations Present in Two Virusesinto One Virus can Enhance its ts and att Phenotypes.

Six paired mutations were combined into fourteen double-pair mutantviruses, of which six could be recovered in Vero or C6/36 cells (Table22). All of the individual paired mutations used in double-pair mutantviruses were is on HuH-7 cells, none was is in Vero cells, and for allcombinations at least one mutation pair conferred an att phenotype insuckling mouse brain. Evaluation of four of the double-pair mutantviruses (Table 23) revealed that combining charge-cluster-to-alaninemutation pairs invariably resulted in the acquisition of a is phenotypein Vero cells (4 out of 4 viruses) and often resulted in a loweredshutoff temperature in HuH-7 cells (3 out of 4 viruses). In half of theviruses assayed, combination of charge-cluster-to-alanine mutation pairsresulted in enhanced restriction of replication (10-fold greater thaneither component mutation) in suckling mouse brain (Table 23) and inSCID-HuH-7 mice (Table 24).

Summary.

The major usefulness of the charge-cluster-to-alanine mutations stemsfrom their design: they are located in the DEN4 non-structural generegion and therefore are envisioned as being useful to attenuate DEN4itself as well as antigenic chimeric viruses possessing the DEN4 NS generegion. Furthermore, they are predicted to be phenotypically more stablethan the single-nucleotide substitution mutant viruses such as the 5-FUmutant viruses. Finally, combinations of mutations are envisioned asbeing created in order to fine-tune attenuation and to further stabilizeattenuation phenotypes.

Example 5 Identification and Characterization of DEN4 Mutant VirusesRestricted in Replication in Mosquitoes

Section 1. Identification of Viruses Showing Restriction of Replicationin Mosquitoes.

In Examples 1 and 4, DEN4 mutant viruses were generated through 5-FUmutagenesis and charge-cluster-to-alanine mutagenesis, respectively, inorder to identify mutations that confer ts, sp and att phenotypes.Another highly desirable phenotype of a dengue virus vaccine isrestricted growth in the mosquito host. A dengue virus vaccine candidateshould not be transmissible from humans to mosquitoes in order toprevent both the introduction of a dengue virus into an environment inwhich it is currently not endemic and to prevent the possible loss ofthe attenuation phenotype during prolonged replication in an individualmosquito host. Loss of the attenuation phenotype could also occurfollowing sustained transmission between humans and mosquitoes.Recently, loss of attenuation of a live attenuated poliovirus vaccinewas seen following sustained transmission among humans (CDC 2000 MMWR49:1094).

In the present example, a panel of 1248 DEN4 mutant viruses generatedthrough 5-FU mutagenesis and 32 DEN4 mutant viruses generated throughcharge-cluster-to-alanine mutagenesis were assayed for restricted growthin mosquito cells. This is a useful preliminary assay for restriction invivo, since restriction in cultured mosquito cells is often, though notalways, associated with poor infectivity for mosquitoes (Huang, C. Y. etal. 2000 J Virol 74:3020-8). Mutant viruses that showed restriction inmosquito cells and robust growth in Vero cells (the substrate forvaccine development, as discussed in Example 6) were targeted forfurther characterization.

Generation and Characterization of the 5-1A1 Mutant.

The generation and isolation of the panel of 1248 5-FU mutant virusesand the panel of 32 charge-cluster-to-alanine mutant viruses have beendescribed in Examples 1, 2, and 4. Vero and C6/36 cells were maintainedas described in Example 1.

Each of the 1248 5-FU mutant viruses and 32 charge-cluster-to-alaninemutant viruses was titered in C6/36 cell monolayers in 24-well plates at32° C. and 5% CO₂. After 5 days, plaques were immunostained withanti-DEN4 rabbit polyclonal antibody and counted as described in thepreceding Examples. Mutant viruses were assayed for one of twophenotypes indicating restricted growth in mosquito cells: either sp inC6/36 cells relative to Vero cells or a ≧3.5 log₁₀ PFU/ml decrease intiter between Vero and C6/36 cells at the permissive temperature foreach cell type. Two mutant viruses, one generated by 5-FU mutagenesis(#5) and one generated by charge-cluster-to-alanine mutagenesis(rDEN4-356,357), showed reduced plaque size in C6/36 cells. After threeterminal dilutions, the 5-FU mutant #5, designated 5-1A1, maintained thereduced plaque size phenotype. Additionally, recombinant virusrDEN4-7546, tested for Vero cell adaptation (discussed in detail inExample 6) also showed reduced plaque size in C6/36 (FIG. 10).

The multicycle growth kinetics of both 5-1A1 and the recombinant wildtype rDEN4 in C6/36 cells were determined as described in Example 1.Briefly, cells were infected in triplicate at a multiplicity ofinfection of 0.01 and samples were harvested at 24-hr intervals. Sampleswere flash frozen and titered in a single assay in Vero cell monolayers.

Oral Infection of Mosquitoes.

Aedes aegypti is one of the primary vectors of dengue virus (Gubler, D.J. 1998 Clin Microbiol Rev 11:480-96). This species was reared at 26° C.and 80% relative humidity (RH) with a 16 hr daylight cycle. Adults wereallowed continuous access to a cotton pad soaked in a 10% sucrosesolution. Five to ten day old female Ae. aegypti which had been deprivedof a sugar source for 48 hr were fed a bloodmeal consisting of equalvolumes of washed human red blood cells, 10% sucrose solution, anddengue virus suspension. The infected blood meal was preparedimmediately prior to feeding and offered to mosquitoes in awater-jacketed feeder covered in stretched parafilm and preheated to 38°C. (Rutledge, L. C. et al. 1964 Mosquito News 24:407-419). Mosquitoesthat took a full bloodmeal within 45 min were transferred to a newcontainer by aspirator and maintained as described above. After 21 days,mosquitoes were stored at −20° C. until dissection.

Intrathoracic Inoculation of Mosquitoes.

The large, non-haematophagous mosquito Toxorhynchites splendens is asensitive host for determining the infectivity of dengue virus. Thisspecies was reared at 24° C. and 75% RH with a 12 hr daylight cycle.Larvae and pupae were fed on appropriately sized Aedes larvae; adultswere allowed continuous access to a cotton pad soaked in a 10% sucrosesolution. Groups of one to ten day old adult T. splendens of both sexeswere immobilized by immersion of their container in an icewater bath andinoculated intrathoracically with undiluted virus and serial tenfolddilutions of virus in 1×PBS. Virus was inoculated in a 0.22 μl doseusing a Harvard Apparatus microinjector (Medical Systems Corp, GreenvaleN.Y.) and a calibrated glass needle (technique is a modification of themethod described in Rosen and Gubler, 1974).

Detection of Viral Antigen in Body and Head Tissues byImmunofluorescence Assay (NA).

Head and midgut preparations of Aedes aegypti and head preparations ofToxorhynchites splendens were made on glass slides as described inSumanochitrapon et al. (Sumanochitrapon, W. et al. 1998 Am J Trop MedHyg 58:283-6). Slides were fixed in acetone for 20 min, and placed at 4°C. until processed by IFA. The primary antibody, hyperimmune mouseascites fluid specific for DEN-4 (HMAF), was diluted 1/100 in PBS-Tween20 (0.05%). Slides were incubated at 37° C. in a humid chamber for 30min, and subsequently rinsed in PBS-Tween 20. The secondary antibody,FITC conjugated goat anti-mouse IgG (KPL, Gaithersburg, Md.), wasdiluted 1/200 in PBS-Tween 20 with 0.002% Evan's Blue. Slides wereviewed on an Olympus BX60 microscope. The infectious dose required toinfect 50% of mosquitoes (ID₅₀) was determined by the method of Reed andMuench (Reed, L. J. & Muench, H. 1938 Am J Hyg 27:493-497). For Aedesaegypti infections, two OID₅₀ (oral infectious dose 50) values werecalculated for each virus: the OID₅₀ required to produce an infection inthe midgut, with or without dissemination to the head, and the OID₅₀required to produce disseminated infection. For Tx. splendens one MID₅₀(mosquito infectious dose 50) value was calculated.

Statistical Analysis.

The percentage of mosquitoes infected by different viruses were comparedusing logistic regression analysis (Statview, Abacus Inc.).

Mutations Restricting Growth of DIEN4 in Mosquito Cells but not VeroCells are Rare.

Out of 1280 mutant viruses initially assayed, only two, #5 andrDEN4-356,357, showed reduced plaque size in C6/36 cells and normalplaque size in Vero cells. One additional virus, rDEN4-7546 (describedin Example 6), with reduced plaque size in C6/36 was detected insubsequent assays. Mutant virus #5 was cloned by three successiveterminal dilutions and designated 5-1A1; rDEN4-7546 and rDEN4-356,357had already been twice-terminally diluted when they were tested in C6/36cells. Virus 5-1A1 has been extensively characterized and its phenotypesare described in detail in the following section. rDEN4-356,357 andrDEN4-7546 are envisioned as being characterized in a similar fashion.

Plaque Size and Growth Kinetics of 5-1A1.

5-1A1 replicated to 6.7 log₁₀ PFU/ml in Vero cells with normal plaquesize and replicated to 7.6 log₁₀ PFU/ml in C6/36 cells with small plaquesize (FIG. 6, Table 25). In comparison, wild type DEN4 used as aconcurrent control replicated to 7.3 log₁₀ PFU/ml in Vero cells, 8.3log₁₀ PFU/ml in C6/36 cells, and showed normal plaque size in both celltypes (FIG. 6, Table 25). The growth kinetics of 5-1A1 was compared tothat of wild type DEN4 by infecting C6/36 cells at an MOI of 0.01 andmonitoring the production of infectious virus. The kinetics andmagnitude of replication of 5-1A1 in C6/36 cells was comparable to thatof wild type DEN4 (FIG. 7).

5-1A1 is Restricted in its Ability to Infect Mosquitoes.

5-1A1 was evaluated for its ability to infect Aedes aegypti mosquitoesthrough an artificial bloodmeal (Table 26). In this assay the ability toinfect the midgut of the mosquito and the ability for a midgut infectionto disseminate to the head are measured separately. The oral infectiousdose 50 (OID₅₀) of wild type DEN4 for the midgut was 3.3 log₁₀ PFU; theOID₅₀ of wild type DEN4 for a disseminated infection was 3.9 log₁₀ PFU.In contrast, 5-1A1 never infected 50% of mosquitoes at the doses used.In order to calculate the OID₅₀ for midgut infections by 5-1A1, it wasassumed that at a 10-fold higher dose, 100% of 25 mosquitoes would havebecome infected. Using this assumption, the conservative estimate of theOID₅₀ for midgut infections by 5-1A1 was ≧3.9 log₁₀ PFU. Because 5-1A1produced only 3 disseminated infections, we did not attempt to calculatean OID₅₀ for this category. 5-1A1 was significantly restricted in itsability to infect the midgut relative to wild type DEN4 (logisticregression, N=150, P<0.001). Additionally, 5-1A1 produced very fewdisseminated infections, but because of low numbers this result was notamenable to statistical analysis.

5-1A1 was also significantly restricted in its ability to infect Tx.splendens mosquitoes following intrathoracic inoculation (Table 27). TheMID₅₀ of wild type DEN4 was 2.3 log₁₀ PFU whereas the MID₅₀ of 5-1A1 wasestimated to be >3.0 log₁₀ PFU (logistic regression, N=36, P<0.01).

5-1A1 does not Show a Ts or an Att Phenotype.

5-1A1 was tested for temperature sensitivity in Vero and HuH-7 cells andfor attenuation in suckling mouse brains as described in Example 1. Themutant virus was not temperature sensitive, as defined in Example 1, andwas not attenuated in suckling mouse brain (Table 25).

Identification and Confirmation of the Mutation Responsible for thePhenotype of 5-1A1.

The nucleotide sequence of the entire genome of 5-1A1 was determined asdescribed in Example 1. Sequencing of 5-1A1 revealed three changes fromthe wild type sequence: two translationally-silent point mutations atpositions 7359 and 9047, and one coding point mutation (C to U) atposition 7129 in the NS4B gene which resulted in a proline to leucinesubstitution.

To formally confirm the effect of the C7129U mutation, the mutation wasinserted into the cDNA p4, which has been described in Examples 1, 2,and 3 and in FIG. 4, using Kunkel mutagenesis as described in Examples 1and 3. The mutagenized cDNA was transcribed and transfected as describedin Example 3, and the resulting virus, after two terminal dilutions, wasdesignated rDEN4-7129-1A. Like 5-1A1, rDEN4-7129-1A showed normal plaquesize and titer in Vero cells and reduced plaque size and normal titer inC6/36 cells (Table 25). rDEN4-7129-1A was not ts on either Vero or HuH-7cells and was not att in suckling mouse brain. Additionally,rDEN4-7129-1A did not show the SCID-HuH-7 att phenotype described inExample 3 (Table 25). The ability of rDEN4-7129-1A to infect mosquitoesis envisioned as being tested in both Ae. aegypti and Tx. splendens.

To test the compatibility of the C7129U mutation and the Δ30 deletion,the C7129U mutation was inserted into rDEN4Δ30 using previouslydescribed techniques. The resulting virus, designated rDEN4Δ30-7129, isenvisioned as being tested for the phenotypes listed in Table 25.

In summary, three mutant viruses, 5-1A1, rDEN4-356,357 and rDEN4-7546,showed a particular combination of phenotypes characterized by normalplaque size and replication to high titers in Vero cells and smallplaque size but unrestricted growth in mosquito cells. 5-1A1 was furthercharacterized and lacked temperature sensitivity in either Vero or HuH-7cells and showed normal levels of replication in mouse brain and inSCID-HuH-7 mice and restricted infectivity for both Ae. aegypti and Tx.splendens mosquitoes. In comparison to wild type rDEN4, the 5-1A1 mutanthad one coding mutation: a point mutation (C to U) at nucleotide 7129 inNS4B resulting in a replacement of Pro with Leu. Because 5-1A1 containsonly a single missense mutation, the phenotype of this mutant virus canbe attributed to the effect of the mutation at position 7129. Theseresults indicate that the 7129 mutation is responsible for the phenotypeof decreased infectivity for mosquitoes and is predicted to be useful torestrict replication of vaccine candidates in mosquitoes. To formallyconfirm this, we have inserted the 7129 mutation into a recombinant DEN4virus. The resulting virus, designated rDEN4-7129-1A, shows an absenceof ts and att phenotypes similar to 5-1A1. It is envisioned as beingtested for mosquito infectivity.

The 7129 mutation is a valuable point mutation to include in a DEN4vaccine candidate and into each of the dengue virus antigenic chimericvaccine candidates since its biological activity is host specific, i.e.,it is restricted in replication in mosquitoes but not in mammals.Moreover, as discussed in Example 6, the 7129 mutation has also beenshown to enhance replication in Vero cells. Thus, its insertion into avaccine candidate is envisioned as enhancing vaccine production intissue culture without affecting the biological properties specified byother attenuating mutations. It is also envisioned as providing a usefulsafeguard against mosquito transmission of a dengue virus vaccine.

Section II. Design of Mutations to Restrict Replication in Mosquitoes

In Section 1 of Example 5, we screened a large panel of mutant virusescarrying both random mutations (generated with 5-fluorouracil) andspecific mutations (generated through charge-cluster-to-alaninemutagenesis) for restricted growth in C6/36 cells, a proxy measure forrestriction in mosquitoes. However, in neither case were mutationsdesigned for the specific purpose of restricting replication inmosquitoes. In this section, we identified nucleotide sequences in the3′ UTR that show conserved differences between the mosquito-transmittedand tick-transmitted flaviviruses. We then altered those sequences inthe DEN4 cDNA p4 by either deleting them altogether or exchanging themwith the homologous sequence of the tick-transmitted Langat virus. Theresulting viruses were assayed for reduced plaque size and titer in bothVero and C6/36 cells and for infectivity for Ae. aegypti and Tx.splendens.

Identification and Modification of Particular 3′ UTR Sequences ShowingConserved Differences Between Vectors.

Several studies (Olsthoorn, R. C. & Bol, J. F. 2001 RNA 7:1370-7;Proutski, V. et al. 1997 Nucleic Acids Res 25:1194-202) have identifiedconserved differences in the nucleotide sequences of the 3′ UTR ofmosquito-transmitted and tick-transmitted flaviviruses. Such differencesare concentrated in the 3′ terminal core region, the approximately 4003′ terminal nucleotides. It has been suggested that these sequences mayhave a vector-specific function (Proutski, V. et al. 1997 Nucleic AcidsRes 25:1194-202). While such a function has not been identified, it maynonetheless be possible to disrupt vector infectivity by deleting orotherwise altering these nucleotides.

To identify target sequences for this type of alteration, we constructedan alignment of the 3′ UTR nucleotide sequences of sevenmosquito-transmitted flaviviruses and four tick-transmitted flaviviruses(FIG. 8). From this alignment, we identified several sequences thatshowed conserved differences between the mosquito-transmittedflaviviruses and tick-transmitted flaviviruses. We then designed primersto alter these sequences in the wt DEN4 cDNA p4 (FIG. 4) in one of twoways: 1) deletion of the nucleotides (A) or 2) replacement of thenucleotides with the homologous sequence from the tick-transmittedflavivirus Langat (swap). Langat was chosen as the template for swappednucleotides because it is naturally attenuated (Pletnev, A. G. 2001Virology 282:288-300), and therefore unlikely to enhance the virulenceof rDEN4 virus derived from the modified cDNA. The DEN4 sequencesaltered and the mutagenesis primers used to do so are listed in Table28. Nucleotides 10508-10530 correspond to the CS2 region identified inprevious studies (Proutski, V. et al. 1997 Nucleic Acids Res25:1194-202).

Mutagenesis of p4, transcription and transfection were conducted aspreviously described in Section I of this Example. All five of theengineered viruses were recovered, and all were subjected to two roundsof terminal dilution as previously described.

Evaluation of Phenotypes: Cell Culture.

Viruses were titered in Vero and C6/36 cells as previously described,and the results are listed in Table 29. All of the viruses replicatedto >5.0 log₁₀ PFU/ml; one of them (rDEN4Δ10508-10530) replicated to >8.0log₁₀ PFU/ml. Only one of the viruses (rDEN4Δ10535-10544) was smallplaque in C6/36 cells; this virus showed wild-type plaque size in Verocells. Interestingly, another virus (rDEN4swap10508-10539) showed wildtype plaque size in C6/36 cells but was sp in Vero cells.

Evaluation of Phenotypes: Mosquito Infectivity.

To date one of the five viruses has been tested for infectivity viaintrathoracic inoculation in Tx. splendens, using previously describedmethods. Virus rDEN4Δ10508-10530 was dramatically restricted ininfectivity relative to the wild type (Table 30). So few mosquitoes wereinfected that it was not possible to calculate an MID₅₀ for this virus.

One of the five viruses has been tested for infectivity of Ae. aegyptifed on an infectious bloodmeal using previously described methods.rDEN4swap10535-10544 (Table 31) caused significantly fewer midgutinfections than wild type rDEN4, but the percentage of disseminatedinfections did not differ between rDEN4swap10535-10544 and wild typerDEN4. All of the viruses are envisioned as being tested for mosquitoinfectivity using both methods.

Summary.

In this example we have outlined two different strategies for preventingmosquito transmission of a dengue vaccine. First, several smallsubstitution mutations, including two point mutations and one pairedcharge-to-alanine substitution, have been shown to restrict thereplication of DEN4 in mosquito C6/36 cells in cell culture, and one ofthese mutations (C7129U) has been shown to restrict the ability of DEN4virus to infect mosquitoes. Second, we have created a variety ofdeletion and substitution mutations in regions of the DEN4 3′ UTR thatshow conserved differences between mosquito-transmitted andtick-transmitted flaviviruses. One of these viruses is sp in C6/36 cellsand at least two of these viruses show some degree of restriction ofmosquito infectivity. By design, the nucleotide sequences in which thesemutations were made are highly conserved among the four dengue serotypesand among mosquito-transmitted flaviviruses in general, indicating thatthey are portable to other vaccine candidates for mosquito-borneflaviviruses. All of the mutations discussed in this Example 11e outsidethe structural genes and so are envisioned as being useful inconstructing antigenic-chimeric vaccine candidates.

Example 6 Adaptation Mutations which Enhance the Replication of DEN4 andDEN4 Chimeric Viruses in Vero Cells

Vero cells are a highly characterized substrate that should be suitablefor the manufacture of live attenuated flavivirus vaccines, such asdengue virus and tick-borne encephalitis virus. In addition, Vero cellscan also be used to grow flaviviruses to high titer for the preparationof an inactivated virus vaccine. Optimal sequences for the efficientgrowth of dengue viruses in Vero cells have not been identified, but itis well known that flaviviruses accumulate mutations during passage invarious cell cultures (Dunster, L. M. et al. 1999 Virology 261:309-18;Theiler, M. & Smith, H. H. 1937 J Exp Med 65:787-800). Inclusion ofspecific sequences in live attenuated viruses that enhance theirreplication in Vero cells and increase the number of doses of vaccineproduced per unit substrate would greatly facilitate their manufacture.Similarly, inclusion of Vero cell growth-promoting sequences in wildtype viruses used for the preparation of an inactivated virus vaccinewould also greatly facilitate the manufacture of the vaccine. Thepresent example identifies mutations that occur following passage ofDEN4 virus and DEN2/4 chimeric viruses in Vero cells. Data derived fromfive sources provided information for this analysis making it possibleto generate a list of Vero cell growth-promoting sequences.

Presence of Identical Mutations in Multiple 5-FU Mutant Viruses.

First, as described in Examples 1 and 2, the genomes of 42 dengue virusclones isolated from a 5-FU mutagenized stock of virus were completelysequenced. If mutations that enhance replication occurred during thepassage of these 42 mutant viruses in Vero cells, then such mutationsshould reveal themselves by representation in more than one clone.Analysis of the 42 sequences revealed the occurrence of specificmissense mutations in coding regions or nucleotide substitutions in UTRsin multiple clones that are not present in the 2A parental virus genome(Tables 11 and 32). These mutations, many of which occur within a 400nucleotide section of the NS4B coding region, represent Verocell-adaptation mutations. One mutation, such as the 4995 mutation,present in eight viruses was found to specify both ts and att phenotypes(Examples 1 and 3). In contrast, the 7163 mutation, present in sixviruses, does not specify a is or att phenotype (Table 13) and thus isan example of a specific Vero cell growth-promoting mutation.

Presence of Vero Cell Adaptation Mutations in Other DEN4 Viruses andDEN2/4 Antigenic Chimeric Viruses.

Second, the 2A-13 dengue virus that was used as a parallel passaged wildtype control during the 5-FU experiments described in Example 1 wasgrown and cloned in Vero cells in the absence of 5-FU in a manneridentical to that of the 5-FU treated viruses. Sequence analysis of this5-FU untreated virus, designated 2A-13-1A1, revealed that the virusgenome contained a mutation at nucleotide 7163 (Example 1 and Table 32),identical to the missense mutation previously identified in 6 of the5-FU mutant viruses (Tables 11 and 32). This indicates that growth andpassage of DEN4 virus in Vero cells is sufficient to acquire thisspecific mutation, i.e. mutagenesis with 5-FU is not required. Thus,information from two separate sources indicates that the 7163 mutationappeared in separate Vero cell passaged viruses, thereby strengtheningthe interpretation that this mutation is growth promoting.

Third, following passage of the 2AΔ30 and rDEN4Δ30 in Vero cells,sequence analysis revealed the presence of a mutation at nucleotides7153 and 7163, respectively. These two mutations were also previouslyidentified among the 5-FU treated viruses (Table 32). Again, identicalmutations appeared following independent passage of virus in Vero cells,corroborating the hypothesis that these mutations confer a growthadvantage in Vero cells.

Fourth, an antigenic chimeric dengue virus vaccine candidate wasgenerated that expressed the structural proteins C, prM, and E from DEN2on a DEN4 wild type genetic background or an attenuated Δ30 geneticbackground. To construct this virus, the C, prM and E region of wildtype cDNA plasmid p4 was replaced with a similar region from DEN2 virusstrain NGC (FIG. 10). Specifically, nucleotides between restrictionsites BglII (nt 88) and XhoI (nt 2345) of p4 were replaced with thosederived from dengue type 2 virus. RNA transcripts synthesized from theresulting p4-D2 plasmid were transfected into Vero cells and rDEN2/4virus was recovered. A further attenuated version of this chimeric viruscontaining the Δ30 mutation, rDEN2/4Δ30, was recovered in C6/36 mosquitocells following transfection of cells with RNA transcripts derivedp4030-D2. However, rDEN2/4Δ30 could not be recovered directly in Verocells. The rDEN2/4Δ30 mutant virus recovered in C6/36 cells replicatedto very low levels in Vero cells (<1.0 log₁₀ PFU/ml) but grew to hightiter in C6/36 cells (>6.0 log₁₀ PFU/ml). Genomic sequence of theC6/36-derived virus matched the predicted cDNA sequence and is shown inAppendix 3. Nevertheless, when C6/36-derived rDEN2/4Δ30 was seriallypassaged 3 to 4 times in Vero cells, a virus population adapted forgrowth in Vero cells emerged. Virus from this Vero cell-adaptedpreparation was cloned and amplified in Vero cells to a titer >6.0 log₁₀PFU/ml. The genomic sequence was determined for 2 independent virusclones and compared to the predicted cDNA sequence (Table 33 and 34).Each cloned virus contains a mutation in a non-structural gene whichcoincides closely in location or sequence with a mutation previouslyidentified among the panel of 5-FU mutagenized viruses. The othermutations in these two clones also might confer a growth advantage inVero cells. Importantly, the mutations identified in Tables 33 and 34are absolutely required for replication in Vero cells, and it would notbe possible to produce the rDEN2/4Δ30 vaccine candidate in Vero cellswithout the growth-promoting mutations identified in Tables 33 and 34.

Fifth, sequence analysis of the dengue 4 wild-type virus strain 814669(GenBank accession no. AF326573) following passage in Vero cellsidentified a mutation in the NS5 region at nucleotide 7630 which hadpreviously been identified among the panel of 5-FU mutagenized viruses(Table 32). This mutation at nucleotide 7630 was introduced intorecombinant virus rDEN4 by site-directed mutagenesis as described inTable 16. The resulting virus, rDEN4-7630, was not temperature sensitivewhen tested at 39° C., indicating that mutation 7630 does not contributeto temperature sensitivity.

Characterization of rDEN2/4Δ30 Chimeric Viruses Containing Single andMultiple Vero Cell Adaptation Mutations.

The generation of chimeric virus rDEN2/4Δ30 provided a uniqueopportunity for evaluating the capacity of individual mutations topromote increased growth in Vero cells. Because rDEN2/4Δ30 replicates tovery low titer in Vero cells, yet can be efficiently generated in C6/36mosquito cells, recombinant virus bearing putative Vero-cell adaptingmutations were first generated in C6/36 cells and then virus titers weredetermined in both C6/36 and Vero cells. As shown in Table 35, additionof a single mutation to rDEN2/4Δ30 resulted in a greater than 1000-foldincrease in titer in Vero cells, confirming the Vero cell adaptationphenotype conferred by these mutations. However, the combination of twoseparate mutations into a single virus did not increase the titer inVero cells beyond the level observed for viruses bearing a singleadaptation mutation. Inclusion of either the 7182 or 7630 mutation inthe cDNA of rDEN2/4Δ30 allowed the virus to be recovered directly inVero cells, circumventing the need to recover the virus in C6/36 cells.

Characterization of the Growth Properties of rDEN4 Viruses ContainingSingle and Multiple Defined Vero Cell Adaptation Mutations.

To confirm the ability of Vero cell adaptation mutations to enhancegrowth of DEN4 viruses, site-directed mutagenesis was used to generaterDEN4 viruses encoding selected individual mutations as described inExamples 1 and 3. Five mutations in NS4B (7153, 7162, 7163, 7182, and7546) from the list of repeated mutations in the 5-FU mutant viruses(Table 32) were introduced singly into the p4 cDNA clone. In addition,the mosquito-restricted, rDEN4-7129 virus was evaluated for enhancedgrowth in Vero cells since the location of this mutation is in the sameregion of NS4B. Each virus, including wild-type rDEN4, was recovered,terminally diluted, and propagated in C6/36 cells to preventintroduction of additional Vero cell adaptation mutations, however,because of its restricted growth in C6/36 cells, rDEN4-7129 waspropagated only in Vero cells.

Plaque size was evaluated for each mutant rDEN4 virus in Vero cells andC6/36 cells and compared to wild-type rDEN4. Six-well plates of eachcell were inoculated with dilutions of virus and plaques were visualizedfive days later. Representative plaques are illustrated in FIG. 10 anddemonstrate that the presence of a Vero cell adaptation mutation doesindeed confer increased virus cell to cell spread and growthspecifically in Vero cells. In C6/36 cells, average plaque size wasapproximately 0.50 mm for both wild-type rDEN4 and each mutant virus(except for rDEN4-7546 and rDEN4-7129 which were smaller than wild-type;see Example 5). However, rDEN4 viruses expressing mutation 7162, 7163,7182, and 7129 had a greater than two-fold increase in plaque size inVero cells compared to wild-type rDEN4 virus. A smaller but consistentincrease in plaque size was observed for rDEN4-7153 and rDEN4-7546.

Growth kinetics and virus yield in Vero cells was assessed for the samepanel of rDEN4 viruses. Vero cells were infected at an MOI of 0.01 andsamples were removed daily for 10 days, titered on Vero cells, andplaques were visualized. The results in FIG. 11 indicate that thepresence of a Vero cell adaptation mutation increased the kinetics ofvirus growth, but had only a marginal effect on the peak virus yield. Atday four post-infection, wild-type rDEN4 grew to 5.2 log₁₀ PFU/ml whilethe level of replication in rDEN4-7129-infected cells was 100-foldhigher. The rest of the mutant rDEN4 viruses had an increased yield atday four ranging from 0.9 (rDEN4-7153) to 1.6 (rDEN4-7162 and -7163)log₁₀ PFU/ml. Interestingly, enhanced kinetics of virus growthcorrelated with increased plaque size in Vero cells. The peak virusyield was reached by day 6 post-infection for rDEN4-7129, -7162, -7163,and -7182 while wild-type rDEN4 did not reach peak titer until day 10.However, the peak virus yield was only slightly higher in rDEN4 virusesexpressing Vero cell adaptation mutations.

In an effort to further enhance rDEN4 replication, especially the peakvirus yield, combinations of selected Vero cell adaptation mutationswere introduced into the rDEN4 background. Three viruses with dualmutations were generated: rDEN4-7153-7163, rDEN4-7153-7182, andrDEN4-7546-7630 and tested in a Vero cell time course infection asdescribed above along with rDEN4 and rDEN4-7162 as a positive control(FIG. 12). The viruses expressing combined mutations grew in a nearlyidentical manner to rDEN4-7162 indicating that these selectedcombinations did not enhance the kinetics or peak virus yield.Additional combinations of these and other Vero cell adaptationmutations are envisioned as increasing peak virus yield.

Discussion.

Some of the growth promoting mutations listed in Table 32 are also foundin homologous regions of DEN1, DEN2, and DEN3 and are envisioned asserving to promote the replication of these viruses in Vero cells.Specifically, the growth promoting mutations indicated in Table 32 thatare present in a DEN4 virus are envisioned as being useful forimportation into homologous regions of other flaviviruses, such as DEN1,DEN2 and DEN3. Examples of such conserved regions are shown in Appendix4 and are listed in Table 36. The nucleotides for both mutation 7129 and7182 are conserved in all four dengue virus serotypes. It is alsointeresting to note that mutation 7129 not only increases growth in Verocells (FIG. 10), but it also forms small plaques in mosquito cells (FIG.6, Table 25). Lee et al. previously passaged DEN3 virus in Vero cellsand performed limited sequence analysis of only the structural generegions of the resulting viruses (Lee, E. et al. 1997 Virology232:281-90). From this analysis a menu of Vero adaptation mutations wasassembled. Although none of these mutations correspond to the Veroadaptation mutations identified in this Example, a single mutation atamino acid position 202 in DEN3 corresponds to mutation 1542 identifiedin 5-FU mutant virus #1012. The current Example emphasizes theimportance in this type of study of determining the sequence of theentire viral genome.

Vero cell growth optimized viruses are envisioned as having usefulnessin the following areas. First, the yield of a live attenuated vaccinevirus in Vero cells is predicted to be augmented. The live attenuatedvaccine candidate is conveniently a DEN4 or other dengue virus or aDEN1/4, DEN2/4, or DEN3/4 antigenic chimeric virus, or a chimeric virusof another flavivirus based on the DEN4 background. The increased yieldof vaccine virus is envisioned as decreasing the cost of vaccinemanufacture. Second, Vero cell adaptation mutations that are attenuatingmutations, such as the 4995 mutation, are envisioned as being stableduring the multiple passage and amplification of virus in Vero cellcultures that is required for production of a large number of vaccinedoses. Third, Vero cell adaptation mutations are actually required forthe growth of the rDEN2/4Δ30 vaccine candidate in Vero cells. Fourth,the increase in yield of a DEN wild type or an attenuated virus isenvisioned as making it economically feasible to manufacture aninactivated virus vaccine: Fifth, the presence of the Vero cell growthpromoting mutations in the DEN4 vector of the rDEN1/4, rDEN2/4, andrDEN3/4 antigenic chimeric viruses or other flavivirus chimeric virusesbased on DEN4 is envisioned as permitting the viruses to grow to a hightiter and as thereby being useful in the manufacture of a inactivatedvirus vaccine. Sixth, the insertion of Vero cell growth promotingmutations into cDNAs such as rDEN2/4Δ30 is envisioned as permittingrecovery of virus directly in Vero cells, for which there are qualifiedmaster cell banks for manufacture, rather than in C6/36 cells for whichqualified cell banks are not available. And seventh, insertion of the7129 and 7182 mutations into DEN1, DEN2, or DEN3 wt viruses isenvisioned as increasing their ability to replicate efficiently and berecovered from cDNA in Vero cells.

Example 7 Assembly of a List of Attenuating Mutations

The data presented in these examples permits the assembly of a list ofattenuating mutations that is summarized in Table 37. This list containsindividual mutations identified in Tables 13-16, 20, and 21 that areknown to independently specify an attenuation phenotype. Mutation 7129is also included since it is derived from virus 5-1A1 shown to beattenuated in mosquitoes. We envision using various combinations ofmutations from this list to generate viruses with sets of desirableproperties such as restricted growth in the liver or in the brain astaught in Example 3 (Table 18) and Example 4 (Tables 23 and 24). Thesemutations are also combinable with other previously describedattenuating mutations such as the Δ30 mutation, as taught in Example 1(Table 6) and Example 3 (Table 19) to produce recombinant viruses thatare satisfactorily attenuated and immunogenic. Mutations listed in Table37 are also envisioned as being combined with other previously describedattenuating mutations such as other deletion mutations or other pointmutations (Blok, J. et al. 1992 Virology 187:573-90; Butrapet, S. et al.2000 J Virol 74:3011-9; Men, R. et al. 1996 Virol 70:3930-7; Puri, B. etal. 1997 J Gen Virol 78:2287-91).

The possibility of importing an attenuating mutation present in oneparamyxovirus into a homologous region of a second paramyxovirus hasrecently been described (Durbin, A. P. et al. 1999 Virology 261:319-30;Skiadopoulos, M. H. et al. 1999 Virology 260:125-35). Such animportation confers an att phenotype to the second virus or,alternatively, further attenuates the virus for growth in vivo.Similarly we envision importing an attenuating mutation present in oneflavivirus to a homologous region of a second flavivirus which wouldconfer an att phenotype to the second flavivirus or, alternatively,would further attenuate the virus for growth in vivo. Specifically, theattenuating mutations indicated in Table 37 are envisioned as beinguseful for importation into homologous regions of other flaviviruses.Examples of such homologous regions are indicated in Appendix 4 for themutations listed in Table 37.

Example 8 Evaluation of Dengue Virus Vaccine in Humans and RhesusMonkeys

The present example evaluates the attenuation for humans and rhesusmonkeys (as an animal model) of a DEN-4 mutant bearing a 30 nucleotidedeletion (Δ30) that was introduced into its 3′ untranslated region bysite-directed mutagenesis and that was found previously to be attenuatedfor rhesus monkeys (Men, R. et al. 1996 J Virol 70:3930-7), asrepresentative of the evaluation of any dengue virus vaccine forattenuation in humans and rhesus monkeys (as an animal model).

Viruses and Cells.

The wild type (wt) DEN-4 virus strain 814669 (Dominica, 1981),originally isolated in Aedes pseudoscutellaris (AP61) cells, waspreviously plaque-purified in LLC-MK2 cells and amplified in C6/36 cellsas described (Mackow, E. et al. 1987 Virology 159:217-28). For furtheramplification, the C6/36 suspension was passaged 2 times in Vero (WHO)cells maintained in MEM-E (Life Technologies, Grand Island, N.Y.)supplemented with 10% FBS. Viruses derived from RNA transfection or usedfor clinical lot development were grown in Vero (WHO) cells maintainedin serum-free media, VP-SFM (Life Technologies).

Construction of DEN-4 Deletion Mutants.

A 30 nucleotide (nt) deletion was previously introduced into the 3′untranslated region of the 2A cDNA clone of wt DEN-4 strain 814669 asdescribed (Men, R. et al. 1996 J Virol 70:3930-7). This deletion removesnucleotides 10478-10507, and was originally designated 3'd 172-143,signifying the location of the deletion relative to the 3′ end of theviral genome. In the current example, this deletion is referred to asΔ30. The full-length 2A cDNA clone has undergone several subsequentmodifications to improve its ability to be genetically manipulated. Aspreviously described, a translationally-silent XhoI restriction enzymesite was engineered near the end of the E region at nucleotide 2348 tocreate clone 2A-XhoI (Bray, M. & Lai, C. J. 1991 PNAS USA 88:10342-6).In this example, the viral coding sequence of the 2A-XhoI cDNA clone wasfurther modified using site-directed mutagenesis to create clone p4: aunique BbvCI restriction site was introduced near the C-prM junction(nucleotides 447-452); an extra XbaI restriction site was ablated bymutation of nucleotide 7730; and a unique SacII restriction site wascreated in the NS5 region (nucleotides 9318-9320). Each of theseengineered mutations is translationally silent and does not change theamino acid sequence of the viral polypeptide. Also, several mutationswere made in the vector region of clone p4 to introduce or ablateadditional restriction sites. The cDNA clone p4Δ30 was generated byintroducing the Δ30 mutation into clone p4. This was accomplished byreplacing the MluI-KpnI fragment of p4 (nucleotides 10403-10654) withthat derived from plasmid 2AΔ30 containing the 30 nucleotide deletion.The cDNA clones p4 and p4Δ30 were subsequently used to generaterecombinant viruses rDEN4 and rDEN4Δ30, respectively.

Generation of Viruses.

Full-length RNA transcripts were synthesized from cDNA clones 2A and2AΔ30 using SP6 RNA polymerase as previously described (Lai, C. J. etal. 1991 PNAS USA 88:5139-43; Men, R. et al. 1996 J Virol 70:3930-7).The reaction to generate full-length RNA transcripts from cDNA clones p4and p4Δ30 was modified and consisted of a 50 μA reaction mixturecontaining 1 μg linearized plasmid, 60 U SP6 polymerase (New EnglandBiolabs (NEB), Beverly, Mass.), 1×RNA polymerase buffer (40 mM Tris-HCl,pH 7.9, 6 mM MgCl₂, 2 mM spermidine, 10 mM dithiothreitol), 0.5 mMm7G(5′)ppp(5′)G cap analog (NEB), 1 mM each nucleotide triphosphate, 1 Upyrophosphatase (NEB), and 80 U RNAse inhibitor (Roche, Indianapolis,Ind.). This reaction mixture was incubated at 40° C. for 90 min and theresulting transcripts were purified using RNeasy mini kit (Qiagen,Valencia, Calif.). For transfection of Vero cells, purified transcripts(1 μg) were mixed with 12 μl DOTAP liposome reagent (Roche) in salinecontaining 20 mM HEPES buffer (pH 7.6) and added to cell monolayercultures in a 6-well plate. After 5-17 days, tissue culture medium washarvested, clarified by centrifugation, and virus was amplified in Verocells. The presence of virus was confirmed by plaque titration. Itshould be noted that during the course of transfection and amplificationof 2AΔ30 to create the vaccine lot, the virus underwent a total of 6passages entirely in Vero cells. The remaining viruses, rDEN4 andrDEN4Δ30 were passaged 5 times in Vero cells to generate the virussuspension used for sequence analysis and studies in rhesus monkeys.

Vaccine Production.

An aliquot of clarified tissue culture fluid containing vaccinecandidate 2AΔ30 was submitted to DynCorp (Rockville, Md.) foramplification of virus in Vero cells and production of a vaccine lot.For vaccine production, 2AΔ30 infected tissue culture supernatant washarvested, SPG buffer added (final concentration: 218 mM sucrose, 6 mML-glutamic acid, 3.8 mM potassium phosphate, monobasic, and 7.2 mMpotassium phosphate, dibasic, pH 7.2), and the virus suspension wasclarified by low speed centrifugation. To degrade residual Vero cellDNA, the vaccine suspension was treated with Benzonase endonuclease(American International Chemical, Natick, Mass.), 100 U/ml and incubatedfor 1 hr at 37° C., followed by high-speed centrifugation (17,000×g, 16hr). The resulting virus pellet was gently rinsed with MEM-E,resuspended in MEM-E containing SPG, sonicated, distributed intoheat-sealed ampoules, and stored frozen at −70° C. Final containersafety testing confirmed microbial sterility, tissue culture purity, andanimal safety. The 2AΔ30 vaccine lot (designated DEN4-9) has a titer of7.48 log 10 PFU/ml, with a single dose of 5.0 log 10 PFU/ml containing<1 pg/ml Vero cell DNA and <0.001 U/ml Benzonase endonuclease.

Sequence of cDNA Clones and Viral Genomes.

The nucleotide sequence of the viral genome region of cDNA plasmids 2Aand p4 was determined on a 310 genetic analyzer (Applied Biosystems,Foster City, Calif.) using vector-specific and DEN-4-specific primers inBigDye terminator cycle sequencing reactions (Applied Biosystems). Thenucleotide sequence of the genomes of the parental wt DEN-4 strain814669 and of recombinant viruses 2A wt, 2AΔ30 (vaccine lot), rDEN4, andrDEN4Δ30 was also determined. Viral RNA was extracted from viruspreparations and serum samples using the QIAamp Viral RNA mini kit(Qiagen). Reverse transcription (RT) was performed using random hexamersand the SuperScript First-Strand Synthesis System for RT-PCR (LifeTechnologies). Overlapping PCR fragments of approximately 2000 basepairs were generated using optimized DEN-4 specific primers andAdvantage cDNA polymerase (ClonTech, Palo Alto, Calif.). Both strands ofpurified PCR fragments were sequenced directly using dye-terminatorreactions as described above and results were assembled into a consensussequence. To determine the nucleotide sequence of the viral RNA 5′ and3′ regions, the 5′ cap nucleoside of the viral RNA was removed withtobacco acid pyrophosphatase (Epicentre, Madison, Wis.) followed bycircularization of the RNA using RNA ligase (Epicentre). RT-PCR wasperformed as described and a cDNA fragment spanning the ligationjunction was sequenced using DEN-4 specific primers. GenBank accessionnumbers have been assigned as follows (virus: accession number): 814669:AF326573, 2AΔ30: AF326826, rDEN4: AF326825, and rDEN4Δ30: AF326827.

Human Vaccine Recipients.

20 normal healthy adult volunteers were recruited by the Johns HopkinsSchool of Hygiene and Public Health Center for Immunization Research(CIR) located in Baltimore, Md. The clinical protocol was reviewed andapproved by the Joint Committee for Clinical Investigation of the JohnsHopkins University School of Medicine and informed consent was obtainedfrom each volunteer. Volunteers were enrolled in the study if they metthe following inclusion criteria: 18-45 years of age; no history ofchronic illness; a normal physical examination; human immunodeficiencyvirus antibody negative, hepatitis B surface antigen negative, andhepatitis C antibody negative; no stool occult blood; and normal valuesfor complete blood cell count (CBC) with differential, hematocrit,platelet count, serum creatinine, serum aspartate amino transferase(AST), alanine amino transferase (ALT), alkaline phosphatase, bilirubin,prothrombin time (PT), partial thromboplastin time (PTT), andurinalysis. Female volunteers were required to have a negative urinepregnancy test prior to vaccination and on the day of vaccination and toagree to use contraception or abstain from sexual intercourse for theduration of the study. Volunteers also lacked serological evidence ofprior flavivirus infection as defined by hemagglutination-inhibitionantibody titer <1:10 to DEN-1, DEN-2, DEN-3, DEN-4, St. Louisencephalitis virus, Japanese encephalitis virus, or yellow fever virusand a plaque-reduction neutralization titer <1:10 to DEN-4 and yellowfever virus.

Studies in Humans.

Volunteers were immunized in three successive cohorts of four, six, andten volunteers to assess the safety of the vaccine. In this study, anillness was defined as the following: dengue virus infection associatedwith a platelet count of <90,000/mm³; serum ALT >4 times normal; oraltemperature >38° C. for >2 successive days; or headache and/or myalgialasting >2 successive days. Systemic illness was defined as theoccurrence of fever >38° C. for >2 consecutive days, or any 2 of thefollowing for at least two consecutive study days: headache, malaise,anorexia, and myalgia/arthralgia. The trials were conducted betweenOctober and April, a time of low mosquito prevalence, to reduce the riskof transmission of vaccine virus from the volunteers to the community.

On the day of vaccination, vaccine candidate 2AΔ30 was diluted to 5.3log₁₀ PFU/ml in sterile saline for injection, USP, and each volunteerwas injected subcutaneously with a 0.5 ml containing 5.0 log₁₀ PFU ofvaccine into the left deltoid region. Volunteers were given a home diarycard on which they were to record their temperature twice daily for days0-5 post-vaccination. The volunteers returned to the clinic each day forexamination by a physician and their diary cards were reviewed. Theinjection site was evaluated for erythema, induration, and tenderness.Clinical signs and symptoms such as headache, rash, petechiae,lymphadenopathy, hepatomegaly, abdominal tenderness, anorexia, nausea,fatigue, myalgia, arthralgia, eye pain, and photophobia were assesseddaily. Symptoms were graded as mild (no need for treatment or a changein activity), moderate (treatment needed or change in activity noted,yet still able to continue daily activity) or severe (confined to bed).Blood was drawn for CBC with differential and for virus quantitation ondays 0, 2 and 4. Volunteers were admitted to the inpatient unit at theCIR on the sixth day after immunization. The study physician evaluatedall volunteers each day by physical examination and interview. Thevolunteers had their blood pressure, pulse, and temperature recordedfour times a day. Blood was drawn each day for CBC with differential andfor virus quantitation and every other day for ALT measurement.Volunteers were confined to the inpatient unit until discharge on studyday 15. On study days 28 and 42, volunteers returned for physicalexamination and blood was drawn for virus quantitation (day 28) and forserum antibody measurement (day 28 and 42).

Virus Quantitation and Amplification.

Serum was obtained for detection of viremia and titration of virus inpositive specimens. For these purposes 8.5 ml of blood was collected ina serum separator tube and incubated at room temperature for less than30 min. Serum was decanted into 0.5 ml aliquots, rapidly frozen in a dryice/ethanol bath and stored at −70° C. Serum aliquots were thawed andserial 10-fold dilutions were inoculated onto Vero cell monolayercultures in 24-well plates. After one hour incubation at roomtemperature, the monolayers were overlaid with 0.8% methylcellulose inOptiMEM (Life Technologies) supplemented with 5% fetal bovine serum(FBS). Following incubation at 37° C. for four days, virus plaques werevisualized by immunoperoxidase staining. Briefly, cell monolayers werefixed in 80% methanol for 30 min and rinsed with antibody buffer (5%nonfat milk in phosphate buffered saline). Rabbit polyclonal DEN-4antibodies were diluted 1:1000 in antibody buffer and added to each wellfollowed by a one hr incubation at 37° C. Primary antibody was removedand the cell monolayers were washed twice with antibody buffer.Peroxidase-labelled goat-anti-rabbit IgG (KPL, Gaithersburg, Md.) wasdiluted 1:500 in antibody buffer and added to each well followed by aone hr incubation at 37° C. Secondary antibody was removed and the wellswere washed twice with phosphate buffered saline. Peroxidase substrate(4 chloro-1-napthol in H₂O₂) was added to each well and visible plaqueswere counted.

For amplification of virus in serum samples, a 0.3 ml aliquot of serumwas inoculated directly onto a single well of a 6-well plate of Verocell monolayers and incubated at 37° C. for 7 days. Cell culture fluidwas then assayed for virus by plaque assay as described above.

Serology.

Hemagglutination-inhibition (HAI) assays were performed as previouslydescribed (Clarke, D. H. & Casals, J. 1958 Am J Trop Med Hyg 7:561-73).Plaque-reduction neutralization titers (PRNT) were determined by amodification of the technique described by Russell (Russell, P. K. etal. 1967 J Immunol 99:285-90). Briefly, test sera were heat inactivated(56° C. for 30 min) and serial 2-fold dilutions beginning at 1:10 weremade in OptiMEM supplemented with 0.25% human serum albumin. rDEN4Δ30virus, diluted to a final concentration of 1000 PFU/ml in the samediluent, was added to equal volumes of the diluted serum and mixed well.The virus/serum mixture was incubated at 37° C. for 30 min. Cell culturemedium was removed from 90% confluent monolayer cultures of Vero cellson 24-well plates and 50 μl of virus/serum mixture was transferred ontoduplicate cell monolayers. Cell monolayers were incubated for 60 min at37° C. and overlaid with 0.8% methylcellulose in OptiMEM supplementedwith 2% FBS. Samples were incubated at 37° C. for 4 days after whichplaques were visualized by immunoperoxidase staining as described above,and a 60% plaque-reduction neutralization titer was calculated.

Studies in Rhesus Monkeys.

Evaluation of the replication and immunogenicity of wt virus 814669, andrecombinant viruses 2A wt, 2AΔ30 (vaccine lot), rDEN4, and rDEN4Δ30 injuvenile rhesus monkeys was performed as previously described (Men R. etal. 1996 J Virol 70:3930-7). Briefly, dengue virus seronegative monkeyswere injected subcutaneously with 5.0 log₁₀ PFU of virus diluted in L-15medium (Quality Biological, Gaithersburg, Md.) containing SPG buffer. Adose of 1 ml was divided between two injections in each side of theupper shoulder area. Monkeys were observed daily and blood was collectedon days 0-10 and 28, and processed for serum, which was stored frozen at−70° C. Titer of virus in serum samples was determined by plaque assayon Vero cells as described above. Neutralizing antibody titers weredetermined for the day 28 serum samples as described above. A group ofmonkeys inoculated with either 2AΔ30 (n=4) or wt virus 814669 (n=8) werechallenged on day 42 with a single dose of 5.0 log₁₀ PFU/ml wt virus814669 and blood was collected for 10 days. Husbandry and care of rhesusmonkeys was in accordance with the National Institutes of Healthguidelines for the humane use of laboratory animals.

Construction and Characterization of DEN-4 Wild Type and Deletion MutantViruses.

The nucleotide and deduced amino acid sequences of the previouslydescribed wt 814669 virus, the DEN-4 2A wt virus derived from it(designated 2A wt), and the 2AΔ30 vaccine candidate derived from 2A wtvirus were first determined. Sequence analysis showed that the wt 814669virus used in this study had apparently accumulated 2 missense mutations(nucleotides 5826 and 7630) and 3 silent mutations during its passageand amplification since these mutations were not described in previouslypublished reports of the viral sequence (GenBank accession numberM14931) and were not present in the 2A cDNA derived from the virus.Sequence comparison between viruses 2A wt and vaccine lot 2AΔ30 revealedthat 2AΔ30 accumulated 2 missense mutations (nucleotides 7153 and 8308)and also confirmed the presence of the Δ30 mutation (nucleotides10478-10507) as well as an additional deletion of nucleotide 10475,which occurred during the original construction of the Δ30 mutation(Men, R. et al. 1996 J Virol 70:3930-7). This sequence analysis revealedsignificant sequence divergence between the biologically-derived wt814669 virus and its recombinant 2A wt derivative and between the 2A wtand 2AΔ30 virus. Since the 2A wt and 2AΔ30 viruses differed atnucleotides other than the deletion mutation, the attenuation phenotypepreviously reported for 2AΔ30 (Men, R. et al. 1996 J Virol 70:3930-7)could not be formally ascribed solely to the Δ30 mutation and may havebeen specified by the mutations at nucleotides 7153, 8308, 10475, or the030 deletion.

To determine whether the Δ30 mutation was responsible for the observedattenuation of 2AΔ30, a second pair of viruses, one with and one withoutthe Δ30 mutation, were produced for evaluation in monkeys. A new DEN-4cDNA vector construct, designated p4, was derived from the 2A-XhoI cDNAclone and translationally-silent mutations were introduced to add orablate several restriction enzyme sites. These sites were added tofacilitate the future genetic manipulation of this DEN-4 wt cDNA by theintroduction of other attenuating mutations if needed. The sequence ofthe genomic region of the p4 cDNA plasmid was identical to that of the2A wt virus except for the engineered restriction site changes and apoint mutation at nucleotide 2440 which was introduced during theoriginal mutagenesis of the 2A cDNA plasmid to create the XhoI site(Bray, M. & Lai, C. J. 1991 PNAS USA 88:10342-6). The Δ30 mutation andthe neighboring deletion at nucleotide 10475 were co-introduced into thep4 plasmid by replacing a short restriction fragment with one derivedfrom the cDNA clone of 2AΔ30. RNA transcripts derived from the p4 cDNAclone and from its Δ30 derivative each yielded virus (designated rDEN4wt and rDEN4Δ30, respectively) following transfection of Vero cells.Sequence analysis of the rDEN4 virus revealed that during its passageand amplification in Vero cells it accumulated 2 missense mutations(nucleotides 4353 and 6195), a silent mutation (nucleotide 10157), and apoint mutation in the 3′ untranslated region (nucleotide 10452). Inaddition to containing the Δ30 and the accompanying deletion atnucleotide 10475, rDEN4Δ30 had also accumulated a missense mutation(nucleotide 7163) and a silent mutation (nucleotide 7295).

Parental wt 814669 virus and recombinant viruses 2A wt, 2AΔ30, rDEN4,and rDEN4Δ30 each replicate in Vero cells to a titer exceeding 7.0 log₁₀PFU/ml, and their replication is not temperature sensitive at 39° C.

Virus Replication, Immunogenicity, and Efficacy in Monkeys.

Groups of rhesus monkeys were inoculated with wt DEN-4 814669, 2A wt,rDEN4, 2AΔ30 and rDEN4Δ30 to assess the level of restriction ofreplication specified by the Δ30 mutation. Serum samples were collecteddaily and titer of virus present in the serum was determined by plaqueenumeration on Vero cell monolayer cultures. Monkeys inoculated with wt814669 virus or its recombinant counterparts, 2A wt or rDEN4, wereviremic for 3 to 4 days with a mean peak virus titer of nearly 2 log₁₀PFU/ml. Monkeys inoculated with virus 2AΔ30 or rDEN4Δ30 had a lowerfrequency of viremia (83% and 50%, respectively), were viremic for onlyabout 1 day, and the mean peak titer was 10-fold lower. Monkeysinoculated with DEN-4 814669, 2A wt, or rDEN4 viruses developed highlevels of neutralizing antibody, with mean titers between 442 and 532,consistent with their presumed wild type phenotype. Monkeys inoculatedwith 2AΔ30 or rDEN4Δ30 developed a lower level of neutralizing antibody,with mean titers of 198 and 223, respectively. The decrease inneutralizing antibody titer in response to 2AΔ30 and rDEN4Δ30 isconsistent with the attenuation phenotype of these viruses. Monkeysinoculated with either 2AΔ30 (n=4) or wt 814669 virus (n=8) werechallenged after 42 days with wt virus 814669. Dengue virus was notdetected in any serum sample collected for up to 10 days following viruschallenge, indicating that these monkeys were completely protectedfollowing immunization with either wt virus or vaccine candidate 2AΔ30.

Since DEN-4 814669, 2A wt, and rDEN4 each manifest the same level ofreplication and immunogenicity in rhesus monkeys, it is reasonable toconclude that the identified sequence differences between thesepresumptive wild type viruses that arose during passage in tissueculture or during plasmid construction do not significantly affect theirlevel of replication in vivo. Similarly, the comparable level ofattenuation of 2AΔ30 and rDEN4Δ30 indicates that the mutations shared bythese viruses, namely, the Δ30 mutation and its accompanying 10475deletion mutation, are probably responsible for the attenuation of theseviruses rather than their incidental sequence differences.

Clinical Response to Immunization with 2AΔ30.

The 2AΔ30 vaccine candidate was administered subcutaneously at a dose of10⁵ PFU to 20 seronegative volunteers. Each of the vaccines was infectedand the virus was well tolerated by all vaccines. Viremia was detectedin 70% of the vaccines, was present only at low titer, and did notextend beyond day 11.

None of the 20 vaccines reported soreness or swelling at the injectionsite. Mild erythema (1-3 mm) around the injection site was noted onexamination of 8 volunteers 30 minutes post-vaccination which resolvedby the next day in 7 of those volunteers and by the third day in theremaining volunteer. Mild tenderness to pressure at the vaccination sitewas noted in 2 volunteers and lasted a maximum of 48 hours. Duringphysical examination, ten volunteers (50%) were noted to a have a verymild dengue-like erythematous macular rash (truncal distribution) whichoccurred with greatest frequency on day 10. None of the volunteers notedthe rash themselves, and it was asymptomatic in each instance. Rash wasseen only in vaccines with detectable viremia. Volunteers did notdevelop systemic illness. Seven volunteers noted an occasional headachethat was described as mild, lasting less than 2 hours, and was notpresent in any volunteer on two consecutive days. One volunteer reportedfever of 38.6° C. and 38.2° C. without accompanying headache, chills,eye pain, photophobia, anorexia, myalgia, or arthralgia as an outpatientthe evening of day 3 and day 5, respectively. However, this volunteerwas afebrile when evaluated by the study staff on the morning of days 3,4, 5 and 6. All other temperature measurements recorded by the volunteeror study staff were normal. Although tourniquet tests were notperformed, two volunteers were noted to have petechiae at the site ofthe blood pressure cuff after a blood pressure measurement was performed(one on day 6, the other on days 7 and 10). Both of these volunteers hadnormal platelet counts at that time and throughout the study.

Significant hematological abnormalities were not seen in any vaccinee.Three vaccines with presumed benign ethnic neutropenia manifested anabsolute neutrophil count (ANC) below 1500/mm³. These three volunteershad baseline ANCs which were significantly lower than the remaining 17volunteers and which did not decrease disproportionately to the othervolunteers. Two of the three volunteers who became neutropenic never haddetectable viremia. A mild increase in ALT levels was noted in 4volunteers, and a more significant increase in ALT level (up to 238IU/L) was noted in one volunteer. These ALT elevations were transient,were not associated with hepatomegaly, and were completely asymptomaticin each of the 5 volunteers. Elevated ALT values returned to normal byday 26 post-vaccination. The volunteer with the high ALT value was alsonoted to have an accompanying mild elevation in AST on day 14 (10⁴ IU/L)which also returned to baseline by day 26 post-vaccination. Thisvolunteer did not have an associated increase in LDH, bilirubin, oralkaline phosphatase levels.

Serologic Response of Humans to Immunization with 2AΔ30.

Each of the twenty vaccines developed a significant rise in serumneutralizing antibody titer against DEN-4 by day 28. The level of serumneutralizing antibody was similar in viremic (1:662) and non-viremicvaccines (1:426). The DEN-4 neutralizing antibody titers of both groupshad not changed significantly by day 42.

Genetic Stability of the Δ30 Mutation.

RT-PCR and sequence analysis of viral RNA isolated from serum samples(n=6) collected from volunteers 6 to 10 days post-vaccination confirmedthe presence of the Δ30 mutation and neighboring deletion at nucleotide10475.

Example 9 Pharmaceutical Compositions

Live attenuated dengue virus vaccines, using replicated virus of theinvention, are used for preventing or treating dengue virus infection.Additionally, inactivated dengue virus vaccines are provided byinactivating virus of the invention using known methods, such as, butnot limited to, formalin or β-propiolactone treatment. Live attenuatedor inactivated viruses containing the mutations described above form thebasis of an improved vaccine for the prevention or treatment of dengueinfection in humans.

Pharmaceutical compositions of the present invention comprise liveattenuated or inactivated dengue viruses, optionally further comprisingsterile aqueous or non-aqueous solutions, suspensions, and emulsions.The composition can further comprise auxiliary agents or excipients, asknown in the art. See, e.g., Berkow et al. eds. 1987 The Merck Manual,15th edition, Merck and Co., Rahway, N.J.; Goodman et al. eds. 1990Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8thedition, Pergamon Press, Inc., Elmsford, N.Y.; Avery's Drug Treatment:Principles and Practice of Clinical Pharmacology and Therapeutics, 3rdedition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. 1987;Osol, A. ed. 1980 Remington's Pharmaceutical Sciences Mack PublishingCo, Easton, Pa. pp. 1324-1341; Katzung, ed. 1992 Basic and ClinicalPharmacology Fifth Edition, Appleton and Lange, Norwalk, Conn.

A virus vaccine composition of the present invention can comprise fromabout 10²-10⁹ plaque forming units (PFU)/ml, or any range or valuetherein, where the virus is attenuated. A vaccine composition comprisingan inactivated virus can comprise an amount of virus corresponding toabout 0.1 to 50 μg of E protein/ml, or any range or value therein.

The agents may be administered using techniques well known to those inthe art. Preferably, agents are formulated and administeredsystemically. Suitable routes may include oral, rectal, transmucosal, orintestinal administration; parenteral delivery, including intramuscular,subcutaneous, intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intradermal, intranasal,or intraocular injections, just to name a few. For injection, the agentsof the invention may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as saline, phosphate bufferedsaline, Tris buffered saline, Hank's buffered saline, growth media suchas Eagle's Minimum Essential Medium (MEM), and the like.

When a vaccine composition of the present invention is used foradministration to an individual, it can further comprise salts, buffers,adjuvants, or other substances which are desirable for improving theefficacy of the composition. Adjuvants useful with the inventioninclude, but are not limited to: (1) aluminum salts (alum), such asaluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2)oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides or bacterial cell wallcomponents), such as for example (a) MF59 (International Publication No.WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85(optionally containing various amounts of MTP-PE, although not required)formulated into submicron particles using a microfluidizer such as Model110Y microfluidizer (Microfluidics, Newton, Mass.), (b) SAF, containing10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, andthr-MDP either microfluidized into a submicron emulsion or vortexed togenerate a larger particle size emulsion, and (c) Ribi™ adjuvant system(RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2%Tween 80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponinadjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.)may be used or particle generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) andIncomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins(IL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc.; (6) mucosal adjuvants such as those derivedfrom cholera toxin (CT), pertussis toxin (PT), E. coli heat labile toxin(LT), and mutants thereof (see, e.g., International Publication Nos. WO95/17211, WO 93/13202, and WO 97/02348); and (7) other substances thatact as immunostimulating agents to enhance the effectiveness of thecomposition.

The pharmacologically active compounds of this invention can beprocessed in accordance with conventional methods of galenic pharmacy toproduce medicinal agents for administration to patients, e.g., mammalsincluding humans.

The compounds of this invention can be employed in admixture withconventional excipients, i.e., pharmaceutically acceptable organic orinorganic carrier substances suitable for parenteral, enteral (e.g.,oral) or topical application, which do not deleteriously react with theactive compounds. Suitable pharmaceutically acceptable carriers includebut are not limited to water, salt solutions, alcohols, gum arabic,vegetable oils, benzyl alcohols, polyethylene glycols, gelatin,carbohydrates such as lactose, amylose or starch, magnesium stearate,talc, silicic acid, viscous paraffin, perfume oil, fatty acidmonoglycerides and diglycerides, pentaerythritol fatty acid esters,hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceuticalpreparations can be sterilized and if desired mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, coloring,flavoring and/or aromatic substances and the like which do notdeleteriously react with the active compounds. They can also be combinedwhere desired with other active agents, e.g., vitamins.

For parenteral application, particularly suitable are injectable,sterile solutions, preferably oily or aqueous solutions, as well assuspensions, emulsions, or implants, including suppositories. Ampoulesare convenient unit dosages.

For enteral application, particularly suitable are tablets, dragees,liquids, drops, suppositories, or capsules. A syrup, elixir, or the likecan be used wherein a sweetened vehicle is employed.

For topical application, there are employed as non-sprayable forms,viscous to semi-solid or solid forms comprising a carrier compatiblewith topical application and having a dynamic viscosity preferablygreater than water. Suitable formulations include but are not limited tosolutions, suspensions, emulsions, creams, ointments, powders,liniments, salves, aerosols, etc., which are, if desired, sterilized ormixed with auxiliary agents, e.g., preservatives, stabilizers, wettingagents, buffers or salts for influencing osmotic pressure, etc. Fortopical application, also suitable are sprayable aerosol preparationswherein the active ingredient, preferably in combination with a solid orliquid inert carrier material, is packaged in a squeeze bottle or inadmixture with a pressurized volatile, normally gaseous propellant,e.g., a freon.

The vaccine can also contain variable but small quantities of endotoxin,free formaldehyde, and preservative, which have been found safe and notcontributing to the reactogenicity of the vaccines for humans.

Example 10 Pharmaceutical Purposes

The administration of the vaccine composition may be for either a“prophylactic” or “therapeutic” purpose. When provided prophylactically,the compositions are provided before any symptom of dengue viralinfection becomes manifest. The prophylactic administration of thecomposition serves to prevent or attenuate any subsequent infection.When provided therapeutically, the live attenuated or inactivated viralvaccine is provided upon the detection of a symptom of actual infection.The therapeutic administration of the compound(s) serves to attenuateany actual infection. See, e.g., Berkow et al. eds. 1987 The MerckManual, 15th edition, Merck and Co., Rahway, N.J.; Goodman et al. eds.1990 Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8thedition, Pergamon Press, Inc., Elmsford, N.Y.; Avery's Drug Treatment:Principles and Practice of Clinical Pharmacology and Therapeutics, 3rdedition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. 1987;Katzung, ed. 1992 Basic and Clinical Pharmacology, Fifth Edition,Appleton and Lange, Norwalk, Conn.

A live attenuated or inactivated vaccine composition of the presentinvention may thus be provided either before the onset of infection (soas to prevent or attenuate an anticipated infection) or after theinitiation of an actual infection.

The vaccines of the invention can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby liveattenuated or inactivated viruses are combined in a mixture with apharmaceutically acceptable vehicle. A composition is said to be a“pharmacologically acceptable vehicle” if its administration can betolerated by a recipient patient. Suitable vehicles are well known tothose in the art, e.g., in Osol, A. ed. 1980 Remington's PharmaceuticalSciences Mack Publishing Co, Easton, Pa. pp. 1324-1341.

For purposes of administration, a vaccine composition of the presentinvention is administered to a human recipient in a therapeuticallyeffective amount. Such an agent is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. A vaccine composition of the presentinvention is physiologically significant if its presence results in adetectable change in the physiology of a recipient patient thatgenerates a host immune response against at least one dengue serotype,stimulates the production of neutralizing antibodies, or leads toprotection against challenge.

The “protection” provided need not be absolute, i.e., the dengueinfection need not be totally prevented or eradicated, if there is astatistically significant improvement compared with a control populationor set of patients. Protection may be limited to mitigating the severityor rapidity of onset of symptoms of the dengue virus infection.

Example 11 Pharmaceutical Administration

A vaccine of the present invention may confer resistance to one or moredengue serotypes by immunization. In immunization, an live attenuated orinactivated vaccine composition is administered prophylactically,according to a method of the present invention. In another embodiment alive attenuated or inactivated vaccine composition is administeredtherapeutically, according to a different method of the presentinvention.

The present invention thus includes methods for preventing orattenuating infection by at least one dengue serotype. As used herein, avaccine is said to prevent or attenuate a disease if its administrationresults either in the total or partial attenuation (i.e., suppression)of a symptom or condition of the disease, or in the total or partialimmunity of the individual to the disease.

At least one live attenuated or inactivated dengue virus, or compositionthereof, of the present invention may be administered by any means thatachieve the intended purpose, using a pharmaceutical composition aspreviously described.

For example, administration of such a composition may be by variousparenteral routes such as subcutaneous, intravenous, intradermal,intramuscular, intraperitoneal, intranasal, oral or transdermal routes.Parenteral administration can be by bolus injection or by gradualperfusion over time. A preferred mode of using a pharmaceuticalcomposition of the present invention is by intramuscular, intradermal orsubcutaneous application. See, e.g., Berkow et al. eds. 1987 The MerckManual 15th edition, Merck and Co., Rahway, N.J.; Goodman et al. eds.1990 Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8thedition, Pergamon Press, Inc., Elmsford, N.Y.; Avery's Drug Treatment:Principles and Practice of Clinical Pharmacology and Therapeutics, 3rdedition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. 1987;Osol, A. ed. 1980 Remington's Pharmaceutical Sciences, Mack PublishingCo, Easton, Pa. pp. 1324-1341; Katzung, ed. 192 Basic and ClinicalPharmacology, Fifth Edition, Appleton and Lange, Norwalk, Conn.

A typical regimen for preventing, suppressing, or treating a denguevirus related pathology, comprises administration of an effective amountof a vaccine composition as described herein, administered as a singletreatment, or repeated as enhancing or booster dosages, over a period upto and including between one week and about 24 months, or any range orvalue therein.

It will be appreciated that the actual preferred amounts of activecompound in a specific case will vary according to the specific compoundbeing utilized, the compositions formulated, the mode of application,and the particular situs and organism being treated. Dosages for a givenhost can be determined using conventional considerations, e.g., bycustomary comparison of the differential activities of the subjectcompounds and of a known agent, e.g., by means of an appropriate,conventional pharmacological protocol.

The dosage of a live attenuated virus vaccine for a mammalian (e.g.,human) subject can be from about 10³-10⁷ plaque forming units (PFU)/kg,or any range or value therein. The dose of inactivated vaccine can rangefrom about 0.1 to 50 μg of E protein. However, the dosage should be asafe and effective amount as determined by conventional methods, usingexisting vaccines as a starting point.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

TABLE 1 Susceptibility of mice to intracerebral DEN4 infection isage-dependent^(a) Mean virus titer (log₁₀PFU/g brain) ± SE followinginoculation at indicated age (days) Virus 7 14 21 2A-13 >6.0 4.0 ± 0.23.1 ± 0.2 rDEN4 >6.0 3.3 ± 0.4 3.3 ± 0.2 rDEN4Δ30 >6.0 3.6 ± 0.2 2.8 ±0.3 ^(a)Groups of 4 or 5 Swiss Webster mice were inoculatedintracerebrally with 10⁵ PFU virus in a 30 μl inoculum. After 5 days,brains were removed, homogenized and titered in Vero cells. SE =Standard error.

TABLE 2 Temperature-sensitive (ts) and mouse brain attenuation (att)phenotypes of 5-FU mutant DEN4 viruses. Virus replication in sucklingmice^(b) Mean virus titer (log₁₀PFU/ml) Mean titer ± at indicated temp.(° C.) SE Mean log₁₀ Vero cells HuH-7 cells (log₁₀PFU/g reductionPhenotype Virus 35 37 38 39 Δ^(a) 35 37 38 39 Δ n brain) from wt^(d) wt(not ts) 2A-13 7.8 7.7 7.6 7.3 0.5 7.8 7.7 7.4 6.4 1.4 66  6.6 ± 0.1^(c)— rDEN4 6.5 6.4 6.4 6.0 0.5 7.1 6.7 6.0 5.5 1.6 66  6.1 ± 0.1^(c) —rDEN4Δ30 6.3 6.1 6.1 5.7 0.6 6.9 6.3 5.9 4.7 2.2 64  5.6 ± 0.1^(c) 0.5ts in Vero and 695 6.2 6.0 5.2   2.6 ^(e) 3.6 6.5 5.5 3.8 <1.6   >4.9 63.0 ± 0.2 3.2 HuH-7 cells 816 6.8 6.4 5.8 3.9 2.9 7.5 6.2 5.5 3.1 4.4 63.3 ± 0.4 2.9 773 7.4 6.6 6.0 3.1 4.3 7.7 6.1 5.2 3.1 4.6 12 3.7 ± 0.12.6 489 7.3 6.6 6.1 3.3 4.0 7.3 6.7 5.4 3.0 4.3 6 4.5 ± 0.5 2.3 173 7.06.1 3.2 2.9 4.1 7.0 3.2 3.0 2.1 4.9 18 4.7 ± 0.4 2.2 509 6.2 5.8 5.5 3.42.8 6.5 6.1 4.5 <1.6   >4.9 6 4.9 ± 0.3 1.9 938 7.1 6.5 5.6 3.1 4.0 7.26.4 5.6 3.1 4.1 6 5.1 ± 0.2 1.7 1033 6.7 6.0 5.9 4.1 2.6 6.9 5.6 4.7<1.6   >5.3 12 4.7 ± 0.2 1.7 239 7.6 6.8 5.6 3.3 4.3 7.6 6.7 4.7 2.5 5.112 4.7 ± 0.3 1.5 793 6.5 5.8 5.3 4.0 2.5 7.2 6.8 5.6 <1.6   >5.6 6 5.4 ±0.3 1.4 759 7.2 6.9 6.4 4.7 2.5 7.5 6.8 6.3 3.1 4.4 12 5.1 ± 0.1 1.4 7186.1 5.9 5.3 3.5 2.6 7.0 6.5 5.7 1.7 5.3 12 5.0 ± 0.3 1.4 473 6.7 6.3 5.42.0 4.7 7.2 6.7 3.7 1.9 5.3 12 5.1 ± 0.3 1.2 ts in only 686 7.0 6.7 6.76.4 0.6 7.3 6.8 6.4 2.2 5.1 12 2.7 ± 0.2 3.8 HuH-7 cells 967 6.8 6.4 6.45.1 1.7 6.8 6.4 5.4 <1.6   >5.2 6 3.6 ± 0.2 2.9 992 7.3 7.1 6.8 5.9 1.47.4 6.9 5.0 <1.6   >5.8 6 3.8 ± 0.1 2.7 571 6.9 7.0 6.4 4.6 2.3 7.0 6.35.2 <1.6   >5.4 6 4.4 ± 0.4 2.4 605 7.6 7.5 7.1 6.9 0.7 7.8 7.2 6.8<1.6   >6.2 12 4.5 ± 0.4 2.1 631 7.1 6.9 6.8 5.0 2.1 7.3 7.1 6.5<1.6   >5.7 12 4.8 ± 0.3 1.9 1175 7.4 7.1 6.9 5.3 2.1 7.6 6.5 4.7 3.34.3 12 4.7 ± 0.2 1.7 ^(a)Reduction in titer (log₁₀PFU/ml) at 39° C.compared to titer at permissive temperature (35° C.). ^(b)Groups of 6suckling mice were inoculated i.c. with 10⁴ PFU virus in a 30 μlinoculum. Brains were removed 5 days later, homogenized, and titered inVero cells. ^(c)Average of 11 experiments with a total of 64 to 66 miceper group. ^(d)Determined by comparing mean viral titers of miceinoculated with mutant virus and the 2A-13 wt control in the sameexperiment (n = 6 or 12). ^(e)Underlined values indicate a 2.5 or 3.5log₁₀ PFU/ml reduction in titer in Vero cells or HuH-7 cells,respectively, at indicated temp when compared to titer at permissivetemp (35° C.).

TABLE 3 Nucleotide and amino acid differences of the 5-FU mutant viruseswhich are is in both Vero and HuH-7 cells. Mutations in coding regionthat Mutations in UTR or coding region that result do not result in anamino acid in an amino acid substitution substitution Nucleotide Gene/Nucleotide Amino Acid Nucleotide Nucleotide Virus position region changechange^(b) position Gene change 173^(a) 7163 NS4B A > C L2354F 10217 NS5 A > U 7849 NS5 A > U N2583I 8872 NS5 A > G K2924R 239^(a) 4995 NS3U > C S1632P 7511 NS4B G > A 10070  NS5 U > C 473^(a) 4480 NS2B U > CV1460A 7589 NS5 G > A 4995 NS3 U > C S1632P 10070  NS5 U > C 489^(a)4995 NS3 U > C S1632P 2232 E U > C 3737 NS2A C > U 509^(a) 4266 NS2B A >G S1389G none 8092 NS5 A > G E2664G 695  40 5′ UTR U > C n/a 1391 E A >G 1455 E G > U V452F 6106 NS3 A > G E2002G 7546 NS4B C > U A2482V 718 2280 E U > C F727L none 4059 NS2A A > G I1320V 4995 NS3 U > C S1632P7630 NS5 A > G K2510R 8281 NS5 U > C L2727S 759^(a) 4995 NS3 U > CS1632P none 8020 NS5 A > U N2640I 773^(a) 4995 NS3 U > C S1632P none793  1776 E G > A A559T 5771 NS3 U > C 2596 NS1 G > A R832K 7793 NS5 U >A 2677 NS1 A > G D859G 4387 NS2B C > U S1429F 816^(a) 4995 NS3 U > CS1632P 6632 NS4A G > A 7174 NS4B C > U A2358V 6695 NS4A G > A 938^(a)3442 NS1 A > G E1114G  747 prM U > C 4995 NS3 U > C S1632P 4196 NS2b U >C 10275 3′ UTR A > U n/a 6155 NS3 G > A 1033^(a)  4907 NS3 A > U L1602F 548 prM C > U 8730 NS5 A > C N2877H 9977 NS5 G > A M3292I ^(a)Virusesthat contain mutation(s) resulting in an a.a. substitution in only a NSgene(s) and/or nucleotide substitutions in the UTRs are indicated; i.e.no a.a. substitutions are present in the structural proteins (C-prM-E).^(b)Amino acid position in DEN4 polyprotein beginning with themethionine residue of the C protein (nt 102-104) as residue #1.Wild-type amino acid on left of amino acid position; mutant amino acidon right.

TABLE 4 Nucleotide and amino acid differences of the 5-FU mutant viruseswhich are is in only HuH-7 cells. Mutations in coding region thatMutations in UTR or coding region that result do not result in an aminoacid in an amino acid substitution substitution Nucleotide Gene/Nucleotide Amino Acid Nucleotide Nucleotide Virus position region changechange^(b) position Gene change 571  586 prM U > C V162A 6413 NS4A U > C7163 NS4B A > U L2354F 7947 NS5 G > A G2616R 605  1455 E G > U V452Fnone 7546 NS4B C > U A2482V 631  595 prM A > G K165R 1175 E G > A 6259NS3 U > C V2053A 5174 NS3 A > G 7546 NS4B C > U A2482V 686^(a) 3575 NS2AG > A M1158I 4604 NS3 A > G 4062 NS2A A > G T1321A 7937 NS5 A > G 7163NS4B A > U L2354F 967  2094 E G > C A665P 4616 NS3 C > U 2416 E U > CV772A 7162 NS4B U > C L2354S 7881 NS5 G > A G2594S 992^(a) 5695 NS3 A >G D1865G 3542 NS2A A > G 7162 NS4B U > C L2354S 1175^(a)  7153 NS4B U >C V2351A 6167 NS3 U > C 10186 NS5 U > C I3362T 10184  NS5 G > A 10275 3′UTR A > U n/a ^(a)Viruses that contain mutation(s) resulting in an a.a.substitution in only a NS gene(s) and/or nucleotide substitutions in theUTRs are indicated; i.e. no a.a. substitutions are present in thestructural proteins. ^(b)Amino acid position in DEN4 polyproteinbeginning with the methionine residue of the C protein (nt 102-104) asresidue #1. Wild-type amino acid on left of amino acid position; mutantamino acid on right.

TABLE 5 Mutations which are represented in multiple 5-FU mutant DEN4viruses. Number of viruses Nucleotide Gene/ Nucleotide Amino acid with“sister” position region change change mutations 1455 E G > U val > phe2 4995 NS3 U > C ser > pro 8 7162 NS4B U > C leu > ser 2 7163 NS4B A > Uor C leu > phe 3 7546 NS4B C > U ala > val 3 10275 3′ UTR A > U n/a^(a)2 ^(a)not applicable

TABLE 6 Addition of ts mutation 4995 to rDEN4Δ30 confers a ts phenotypeand further attenuates its replication in suckling mouse brain.Replication in suckling mice^(b) Mean virus titer ± Mean virus titer(log₁₀ PFU/ml) at indicated temp (° C.) SE Mean log₁₀ Vero cells HuH-7cells (log₁₀PFU/g reduction from Virus 35 37 38 39 Δ^(a) 35 37 38 39 Δbrain) wt^(c) 2A-13 7.1 7.1 6.9 6.8 0.3 7.4 7.3 6.7 6.4 1.0 6.5 ± 0.1 —rDEN4 7.0 6.8 6.6 6.4 0.6 7.5 7.3 6.7 6.4 1.1 6.1 ± 0.2 — rDEN4Δ30 7.06.7 6.2 6.2 0.8 7.5 7.0 6.5 5.1 2.4 5.9 ± 0.1 0.2 rDEN4-4995 5.7 4.9 3.6<1.6   >4.1 6.4 5.7 4.0 <1.6   >4.8 3.2 ± 0.2 2.9 rDEN4Δ30- 5.9 4.9 3.9<1.6 ^(d) >4.3 6.4 5.6 4.4 <1.6   >4.8 3.0 ± 0.3 3.1 4995 ^(a)Reductionin titer (log₁₀PFU/ml) at 39° C. compared to titer at permissivetemperature (35° C.). ^(b)Groups of 6 suckling mice were inoculated i.c.with 10⁴ PFU virus in a 30 μl inoculum. Brains were removed 5 dayslater, homogenized, and titered in Vero cells. The limit of detection is2.0 log₁₀PFU/g brain. ^(c)Determined by comparing mean viral titers ofmice inoculated with sample virus and rDEN4 control. ^(d)Underlinedvalues indicate a 2.5 or 3.5 log₁₀PFU/ml reduction in titer in Verocells or HuH-7 cells, respectively, at indicated temperature whencompared to permissive temperature.

TABLE 7 Temperature-sensitive (ts) and mouse brain attenuation (att)phenotypes of 5-FU DEN4 mutant viruses which exhibit a small plaque (sp)phenotype. Phenotype Mean virus titer (log₁₀PFU/ml) at indicated temp (°C.) sp ts Vero cells HuH-7 cells Vero HuH-7 Vero HuH-7 Virus 35 37 38 39Δ^(a) 35 37 38 39 Δ − − − − 2A-13 7.9 7.5 7.7 7.2 0.7 7.9 7.7 7.3 6.91.0 − − − − rDEN4 7.9 7.6 7.7 7.3 0.6 8.1 7.6 7.5 6.7 1.4 − − − −rDEN4Δ30 7.3 6.6 6.6 6.1 1.2 7.3 7.2 6.9 5.9 1.4 + + + + 574 6.6^(x) 5.53.8 <1.6 ^(e)  ≧5.0 6.6^(x) 4.9 5.0 <1.6  ≧5.0 + + + + 1,269 5.3^(x) 4.83.9 <1.6  ≧3.7 4.0^(x) 2.4 2.0 <1.6  ≧2.4 + + + + 1,189 6.3^(x) 5.2 4.53.8 2.5 5.5^(x) 3.7 2.3 <1.6  ≧3.9 + + − − 569 5.8^(x) 5.6 5.6 3.7 2.16.2^(x) 6.0 5.7 5.0 1.2 + + − − 761 5.0^(x) 4.7 4.2 2.7 2.3 5.6^(x) 5.34.5 2.6 3.0 − + + + 506 7.0 6.8 5.6 2.6 4.4 6.7^(x) 4.3 <1.6  2.0 4.7− + + + 1,136 5.1 4.2 2.6 <1.6  ≧3.5 5.7^(x) 3.0 3.0 <1.6  ≧4.1 − + + +1,029 6.9 5.8 5.8 2.9 4.0 7.0^(x) 5.8 5.2 2.5 4.5 − + + + 1,081 6.9 5.84.7 3.9 3.0 5.8^(x) 4.1 3.3 1.9 3.9 − + + + 529 6.9 6.5 5.9 4.0 2.97.1^(x) 5.3 4.4 <1.6  ≧5.5 − + + + 1,114 6.7 6.4 6.2 2.5 4.2 5.7^(x) 3.02.9 1.9 3.8 − + + + 922 7.3 7.2 6.8 3.8 3.5 7.4^(x) 5.3 4.1 3.0 4.4− + + + 311 6.9 5.9 4.3 1.5 5.4 7.1^(x) 5.4 3.6 <1.6  ≧5.5 − + + + 3266.6 5.7 4.5 3.1 3.5 7.0^(x) 5.5 4.1 2.0 5.0 − + − + 1,104 7.1 6.8 6.86.1 1.0 7.2^(x) 6.4 5.8 2.8 4.4 − + − + 952 7.1 7.0 6.7 5.6 1.5 7.3^(x)6.3 5.6 3.0 4.3 − + − + 738 6.5 6.0 5.9 5.7 0.8 6.9^(x) 6.1 5.0 3.1 3.8− + − + 1,083 7.4 7.3 7.4 5.8 1.6 7.4^(x) 6.6 4.5 <1.6  ≧5.8 − + − −1,096 7.5 7.1 6.9 5.5 2.0 7.5^(x) 6.6 5.6 4.8 2.7 − + − − 1,021 7.0 6.96.6 6.3 0.7 6.9^(x) 5.7 4.4 4.0 2.9 − + − − 1,023 6.6 6.4 6.0 5.8 0.86.1^(x) 5.6 4.7 3.3 2.8 − + − − 1,012 7.5 7.1 7.0 5.7 1.8 7.4^(x) 6.86.8 5.6 1.8 Replication in suckling mice^(b) Mean virus Phenotype titer± SE sp ts (log₁₀PFU/g Mean log₁₀ Vero HuH-7 Vero HuH-7 Virus n brain)reduction from wf^(d) − − − − 2A-13 66  6.6 ± 0.1^(c) — − − − − rDEN4 66 6.1 ± 0.1^(c) — − − − − rDEN4Δ30 64  5.6 ± 0.1^(c) 0.5 + + + + 574 62.1 ± 0.1 5.1 + + + + 1,269 6 2.7 ± 0.2 4.1 + + + + 1,189 12 3.2 ± 0.43.7 + + − − 569 12 1.9 ± 0.1 4.6 + + − − 761 12 2.0 ± 0.1 4.2 − + + +506 6 2.2 ± 0.1 4.7 − + + + 1,136 6 2.9 ± 0.3 4.5 − + + + 1,029 6 2.2 ±0.1 4.2 − + + + 1,081 12 2.6 ± 0.2 3.9 − + + + 529 6 3.1 ± 0.7 3.8− + + + 1,114 6 2.7 ± 0.3 3.7 − + + + 922 12 3.5 ± 0.1 2.9 − + + + 31112 6.1 ± 0.3 0.9 − + + + 326 6 6.0 ± 0.1 0.9 − + − + 1,104 6 2.2 ± 0.14.7 − + − + 952 6 2.4 ± 0.3 4.5 − + − + 738 12 4.4 ± 0.4 2.3 − + − +1,083 12 4.5 ± 0.4 2.0 − + − + 1,096 6 2.9 ± 0.2 3.5 − + − + 1,021 6 3.9± 0.6 2.6 − + − − 1,023 12 4.2 ± 0.3 2.3 − + − − 1,012 6 6.1 ± 0.1 0.8^(a)Reduction in mean virus titer (log₁₀PFU/ml) at 39° C. compared topermissive temperature (35° C.). ^(b)Groups of 6 suckling mice wereinoculated i.c. with 10⁴ PFU virus. Brains were removed 5 days later,homogenized, and titered in Vero cells. ^(c)Average of 11 experimentswith a total of 64 to 66 mice per group. ^(d)Determined by comparingmean viral titers of mice inoculated with mutant virus and concurrent2A-13 wild type (wt) virus control (n = 6 or 12). ^(e)Underlined valuesindicate a 2.5 or 3.5 log₁₀PFU/ml reduction in titer in Vero cells orHuH-7 cells, respectively, at indicated temperature when compared topermissive temperature (35° C.). ^(x)Small plaque size at 35° C.; smallplaques have a diameter of <1.0 mm compared to wild type plaque diameterof 1.5-2.0 mm in Vero cells, or a diameter of <0.4 mm compared to wildtype plaque diameter of 0.75 to 1.0 mm in HuH-7 cells.

TABLE 8 Viruses with both ts and sp phenotypes are more restricted inreplication in mouse brain than those with only a ts phenotype. Meanlog₁₀ Cell culture Number reduction in virus phenotype of viruses titerfrom control^(b,c) ts^(a) 20 2.1 ± 0.2 sp 6 3.0 ± 0.6 ts/sp 16 3.5 ± 0.3^(a)20 ts mutant viruses without an sp phenotype were previouslydescribed (Example 1). ^(b)Determined by comparing mean viral titers ofgroups of mice inoculated with mutant virus and concurrent 2A-13parallel-passaged control virus. ^(c)Significant difference between tsgroup and ts/sp group, Tukey-Kramer test (P < 0.05)

TABLE 9 Nucleotide and amino acid differences of the 5-FU mutant DEN4viruses which produce small plaques in both Vero and HuH-7 cells.Mutations in coding regions Mutations in UTR or in coding regions thatthat do not result in an amino result in an amino acid substitution acidsubstitution Nucleotide Gene/ Nucleotide Amino acid NucleotideNucleotide Virus position region change change^(b) position Gene change569 826 prM G > A R242K 1946 E C > U 832 prM C > U P244L 7546 NS4B C > UA2482V 10275 3′ UTR A > U n/a 10279 3′ UTR A > U n/a 574 1455 E G > UV452F 1349 E C > U 1963 E U > C V621A 3880 NS2A A > G K1260R 7546 NS4BC > U A2482V 7615 NS5 A > G N2505S 10413 3′ UTR A > G n/a 761 424 C U >C I108T none 2280 E U > C F727L 7131 NS4B A > G T2344A 7486 NS4B A > GN2462S 1189^(a ) 3303 NS1 A > G R1068G 6719 NS4A U > C 4812 NS3 G > AV1571I 5097 NS3 G > A D1666N 7182 NS4B G > A G2361S 1269 2112 E U > CF671L 542 prM C > U 3256 NS1 G > A G1052E 3993 NS2A U > C F1298L 7183NS4B G > U G2361V ^(a)Virus contains missense mutations in only thenon-structural genes. ^(b)Amino acid position in DEN4 polyproteinbeginning with the methionine residue of the C protein (nt 102-104).Wild type amino acid on left of amino acid position; mutant amino acidon right.

TABLE 10 Nucleotide and amino acid differences of the 5-FU mutant DEN4viruses which produce small plaques in only HuH-7 cells. Mutations incoding regions that Mutations in UTR or in coding regions that result donot result in an amino acid in an amino acid substitution substitutionNucleotide Gene/ Nucleotide Amino acid Nucleotide Nucleotide Virusposition region change change^(b) position Gene change  311 1519 E A > GN473S 6761 NS4A C > U 2305 E G > A R735K 10070 NS5 U > C 4896 NS3 G > UA1599S  326 1587 E C > U P496S 1523 E G > A 7546 NS4B C > U A2482V 6080NS3 U > C 10070 NS5 U > C  506 1455 E G > U V452F 3887 NS2A A > G 1902 EG > A V601M 5789 NS3 G > C 7546 NS4B C > U A2482V 10275 3′ UTR A > U n/a 529 777 prM U > C S226P none 4641 NS3 A > G I1514V 7153 NS4B U > CV2351A 8245 NS5 U > C I2715T 10279 3′ UTR A > C n/a  738^(a) 3540 NS2AG > A E1147K none 7162 NS4B U > C L2354S  922^(a) 4306 NS2B A > G N1402S7736 NS5 G > A 5872 NS3 C > U T1924I 7163 NS4B A > U L2354F 10279 3′ UTRA > C n/a  952 1449 E G > U V450L none 1455 E G > U V452F 7546 NS4B C >U A2482V 7957 NS5 U > C V2619A 9543 NS5 A > G I3148V 1012 1542 E A > GK481E 953 E A > G 7162 NS4B U > C L2354S 1205 E G > A 10542 3′ UTR A > Gn/a 4425 NS2B U > C 1021 2314 E U > C I738T 665 prM C > A 3205 NS1 C > UA1035V 5750 NS3 C > U 4029 NS2A U > C C1310R 9959 NS5 C > U 7163 NS4BA > C L2354F 10275 3′ UTR A > U n/a 10279 3′ UTR A > U n/a 1023 2283 EG > A G728R 1001 E C > U 7182 NS4B G > A G2361S 1958 E A > G 3873 NS2aU > C 8486 NS5 C > U 1029 850 prM C > U A250V 3867 NS2a C > U 3087 NS1A > G T996A 4891 NS3 U > C I1597T 1081^(a) 2650 NS1 A > G N850S 6326 NS3C > U 7163 NS4B A > U L2354F 9146 NS5 C > U 1083^(a) 3702 NS2A G > AA1201T 3353 NS1 A > G 7153 NS4B U > C V2351A 6155 NS3 G > A 10634 3′ UTRU > C n/a 1096 892 prM G > A R264Q 665 prM C > A 7163 NS4B A > C L2354F4427 NS2b G > A 8659 NS5 C > U P2853L 1104 1692 E G > A V531M none 5779NS3 C > U A1893V 7546 NS4B C > U A2482V 1114 709 prM A > G K203R 1076 EU > C 3693 NS2A A > G I1198V 1182 E C > U 4614 NS3 U > C F1505L 5690 NS3C > U 7546 NS4B C > U A2482V 9942 NS5 A > G T3281A 1136^(a) 3771 NS2AA > G R1224G 5621 NS3 A > G 4891 NS3 U > C I1597T 10275 3′ UTR A > U n/a^(a)Viruses that contain missense mutations in only the non-structuralgenes and/or mutations in the UTRs. ^(b)Amino acid position in DEN4polyprotein beginning with the methionine residue of the C protein (nt102-104). Wild type amino acid on left of amino acid position; mutantamino acid on right.

TABLE 11 Putative Vero cell adaptation mutations derived from the fullset of 5-FU mutant viruses. 5-FU mutant viruses No. of virusesNucleotide Gene/region Nucleotide Amino acid with the position (a.a.#)^(b) change change mutation 1455 E (452) G > U Val > Phe 5 2280 E(727) U > C Phe > Leu 2 4891 NS3 (1597) U > C Ile > Thr 2 4995 NS3(1599) U > C Ser > Pro 8 7153 NS4B (2351) U > C Val > Ala 3 7162 NS4B(2354) U > C Leu > Ser 4 7163 NS4B (2354) A > U or C Leu > Phe 7 7182NS4B (2361) G > A Gly > Ser 2 7546 NS4B (2482) C > U Ala > Val 10 7630NS5 (2510) A > G Lys > Arg 1 10275 3′ UTR A > U n/a^(a) 6 10279 3′ UTRA > C n/a 4 ^(a)not applicable ^(b)Amino acid position in DEN4polyprotein beginning with the methionine residue of the C protein (nt102-104) as residue #1.

TABLE 12 Mutagenic oligonucleotides used to generate recombinantDEN4 viruses containing single 5-FU mutations. Recombinant SEQ virusNucleotide Amino acid ID NO. (rDEN4-) change change Gene pUCcloneRE site^(a) Oligonucleotide^(b) 23   40 U > C n/a 5′ UTR pUC-NheI BsaWICAGTTCCAAAcCGGAAGCTTG 24 2650 A > G Asn > Ser NS1 pUC-NS1 BsiWICCAACGAGCTAt cgTAcGTTCTCTGGG 25 3303 A > G Arg > Gly NS1 pUC-NS1 StyIGATTGTGACCATgGcGGCCCATCTTTG 26 3442 A > G Glu > Gly NS1 pUC-NS1 BlpIGGAGATTAGGCCgcTGAGcGgtAAAGAAGAG 27 3540 G > A Glu > Lys NS2A pUC-NS1BsmI GTTTGTGGAAaAATGtcTGAGGAGAA 28 3575 G > A Met > Ile NS2A pUC-NSISspI CTAGGAAACACAT aATATTAGTTGTGG 29 3702 G > A Ala > Thr NS2A pUC-NS2ABglI CAGATCCACCTAaCCATaATGGCAGTG 30 3771 A > G Arg > Gly NS2A pUC-NS2AAvaI GGAAACTCACcTCggGAGAGACAGC 31 4059 A > G Ile > Val NS2A pUC-NS2ABstEII TTGGGTAGAggTcACcGCACTCATCC 32 4062 A > G Thr > Ala NS2A pUC-NS2ABsrBI GTAGAAATAg CcGCtCTCATCCTAG 33 4266 A > G Ser > Gly NS2B pUC-NS2ASnaBI GGCGGCTTACGTaATGgGaGGTAGCTCAGC 34 4306 A > G Asn > Ser NS2BpUC-NS2A AlwNI CTAGAGAAGGCaGCttctGTGCAGTGG 35 4480 U > C Val > Ala NS2BpUC-NS2A MscI CCTTGGCcATTCCAGcaACAATGAC 36 4812 G > A Val > Ile NS3pUC-NS2A ApoI GACGTTCAaaTttTaGCCATAGAACC 37 4891 U > C Ile > Thr NS3pUC-NS2A KasI CTGGAGAAAcgGGcGCcGTAACATTAG 38 4896 G > U Ala > Ser NS3pUC-NS2A BstEII GAAATTGGAtCgGTAACcTTAGATTTC 39 4907 A > U Leu > Phe NS3pUC-NS2A AclI GGAGCAGTAACgTT tGATTTCAAACCC 40 4995 U > C Ser > Pro NS3pUC-NS2A BsaJI GTTACCAAA cCtGGgGATTACGTC 41 5097 G > A Asp > Asn NS3pUC-NS3 BspHI GATTAACTATcATGa ACTTACACCC 42 5695 A > G Asp > Gly NS3pUC-NS3 BanI GGAAAACCTTTGgcACcGAGTATCC 43 5872 C > U Thr > Ile NS3pUC-NS3 BsrFI TCCAGTGAt aCCgGCtAGCGCTGCTC 44 6106 A > G Glu > Gly NS3pUC-NS3 MscI GCCTCAGAGGtGgcCAAAGGAAG 45 6259 U > C Val > Ala NS3 pUC-NS3BglII ACATGGAGGcaGAgATcTGGACTAGA 46 7153 U > C Val > Ala NS4B pUC-NS4AMscI AAAGCATGgCcAAGGATGCTGTC 47 7162 U > C Leu > Ser NS4B pUC-NS4A BlpIGCATAATGGACgctAAGCATGACTAAGG 48 7163 A > C Leu > Phe NS4B pUC-NS4A ApaLITTATTGCATAgTGcACg AAAAGCATG 49 7174 C > U Ala > Val NS4B pUC-NS4A BsaAIGGGCCTATTATTaCgTAATGGAC 50 7182 G > A Gly > Ser NS4B pUC-NS4A n/aCTGCAATCCTGGtgaTATTATTGC 51 7546 C > U Ala > Val NS4B pUC-NS5A AclICTCATAAAGAAcGttCAAACCCT 52 7630 A > G Lys > Arg NS5 pUC-NS5A HgaICATTAGACAGAcgcGAGTTTGAAG 53 7849 A > U Asn > Ile NS5 pUC-NS5A HgaITGGCGACgCTCAAGAtaGTGACTGAAG 54 8020 A > U Asn > Ile NS5 pUC-NS5A ClaIGAGTCATCaTCgAt aCCAACAATAG 55 8092 A > G Glu > Gly NS5 pUC-NS5A EcoRICTTCAAAACCTGgcTTCTGCATCAAAG 56 8281 U > C Leu > Ser NS5 pUC-NS5B XmnICAAAGATGTTGagcAACAGGTTCACAAC 57 8730 A > C Asn > His NS5 pUC-NS5B AvaIGGAAAGAAGAAAcAcCCgAGACTGTGC 58 8872 A > G Lys > Arg NS5 pUC-NS5B PvuIGGGAACTGGTcGAtc gAGAAAGGGC 59 9977 G > A Met > Ile NS5 pUC-NS5C SfcICCAGTGGATtACtACaGAAGATATGCTC 60 10186  U > C Ile > Thr NS5 pUC-NS5C AgeICAGGAACCTGAcCGGtAAAGAGGAATACG 61 10275  A > U n/a 3′ UTR pUC-NS5C n/aCTGTAATTACCAACAtCAAACACCAAAG 62 10279  A > C n/a 3′ UTR pUC-NS5C n/aCCAACAACAAcCACCAAAGGCTATTG 63 10634  U > C n/a 3′ UTR pUC-3′UTR n/aGGATTGGTGTTGTcGATCCAACAGG ^(a)Primers were engineered which introduced(underline) or ablated (hatched line) translationally-silent restrictionenzyme sites. ^(b)Lowercase letters indicate nt changes and bold lettersindicate the site of the 5-FU mutation, which in some oligonucleotidesdiffers from the original nucleotide substitution change in order tocreate a unique restriction enzyme site. The change preserves the codonfor the amino acid substitution.

TABLE 13 sp, ts and mouse attenuation phenotypes of rDEN4 mutant virusesencoding single mutations identified in six sp 5-FU mutant viruses.Replication in suckling mice^(b) Gene/ Mean virus titer (log₁₀PFU/ml) atMean virus Mean log₁₀- 5-FU region indicated temp (° C.) titer ± SE unitreduction mutant containing Vero cells HuH-7 cells (log₁₀PFU/g fromvalue for virus Virus mutation 35 39 Δ^(a) 35 39 Δ n brain) wt^(c) 2A-137.6 7.1 0.5 7.8 6.6 1.2 30 6.5 ± 0.1 — rDEN4 7.6 6.8 0.8 8.0 6.7 1.3 545.8 ± 0.1 — rDEN4Δ30 7.6 6.9 0.7 7.7 5.6 2.1 30 5.6 ± 0.1 0.2 738 parent6.5 5.7 0.8 ^(x)6.9   3.1 ^(e) 3.8 12 4.4 ± 0.4 2.3 rDEN4-3540 NS2A 6.95.1 1.8 7.4 3.7 3.7 12 4.1 ± 0.3 1.7 rDEN4-7162 NS4B 7.2 6.8 0.4 7.4 6.60.8 8 5.6 ± 0.3 0.3 922 parent 7.3 3.8 3.5 ^(x)7.4  3.0 4.4 12 3.5 ± 0.12.9 rDEN4-4306 NS2B ^(x)5.0  2.2 2.8 ^(x)5.6  <1.6  >4.0 12 1.7 ± 0.14.1 rDEN4-5872 NS3 5.7 2.5 3.2 ^(x)6.5  <1.6  >4.9 12 4.5 ± 0.3 1.3rDEN4-7163 NS4B 7.8 7.2 0.6 8.0 7.4 0.6 6 6.2 ± 0.2 (+)0.1   rDEN4-102793′ UTR 6.9 5.7 1.2 7.7 5.7 2.0 6 4.8 ± 0.2 0.7 1081 parent 6.9 3.9 3.0^(x)5.8  1.9 3.9 12 2.6 ± 0.2 3.9 rDEN4-2650 NS1 5.1 3.0 2.1 ^(x)5.5 2.8 2.7 12 3.0 ± 0.3 2.8 rDEN4-7163 NS4B 7.8 7.2 0.6 8.0 7.4 0.6 6 6.2 ±0.2 (+)0.1   1083 parent 7.4 5.8 1.6 ^(x)7.4  <1.6  ≧5.8 12 4.5 ± 0.42.0 rDEN4-3702 NS2A 6.8 5.6 1.2 7.6 4.7 2.9 18 4.9 ± 0.3 0.9 rDEN4-7153NS4B 7.7 7.2 0.5 8.0 6.9 1.1 6 5.7 ± 0.1 0.2 rDEN4-10634 3′ UTR 4.9 1.63.3 ^(x)5.7  <1.6  ≧4.1 12 2.4 ± 0.3 3.4 1136 parent 5.1 <1.6  ≧3.5^(x)5.7  <1.6  >4.1 6 2.9 ± 0.3 4.5 rDEN4-3771 NS2A 7.0 4.6 2.4 ^(x)7.6 3.7 3.9 12 2.6 ± 0.4 3.2 rDEN4-4891 NS3 7.1 <1.6  >5.5 ^(x)7.4 <1.6  >5.8 12 2.5 ± 0.3 3.5 rDEN4-10275 3′ UTR 6.9 5.8 1.1 7.1 5.2 1.9 65.0 ± 0.3 0.5 1189 parent ^(x)6.3  3.8 2.5 ^(x)5.5  <1.6  >3.9 12 3.2 ±0.4 3.7 rDEN4-3303 NS1 6.1 4.8 1.3 6.6 3.9 2.7 8 5.7 ± 0.4 0.2rDEN4-4812 NS3 7.0 6.3 0.7 7.1 6.3 0.8 12 4.8 ± 0.2 1.0 rDEN4-5097 NS3^(x)5.0  <1.6  >3.4 ^(x)4.6  <1.6  >3.0 12 1.8 ± 0.1 4.0 rDEN4-7182 NS4B7.7 6.9 0.8 7.8 6.8 1.0 6 6.2 ± 0.1 (+)0.1   Replication in HuH-7-SCIDmice^(d) Mean peak Gene/region virus titer ± Mean log₁₀-unit 5-FU mutantcontaining SE (log₁₀PFU/ml reduction from virus Virus mutation n serum)value for wt^(c) 2A-13 29 6.8 ± 0.2 — rDEN4 32 6.3 ± 0.2 — rDEN4Δ30 185.4 ± 0.2 0.9 738 parent 9 5.4 ± 0.7 1.9 rDEN4-3540 NS2A 5 6.1 ± 0.3(+)0.1   rDEN4-7162 NS4B 5 6.8 ± 0.6 0.3 922 parent 6 6.2 ± 0.2 0.4rDEN4-4306 NS2B 5 5.2 ± 0.6 1.1 rDEN4-5872 NS3 5 6.2 ± 0.5 0.1rDEN4-7163 NS4B 6 5.8 ± 0.6 (+)0.2   rDEN4-10279 3′ UTR 4 6.7 ± 0.2 0.41081 parent 4 4.2 ± 0.5 2.4 rDEN4-2650 NS1 6 4.7 ± 0.5 2.2 rDEN4-7163NS4B 6 5.8 ± 0.6 (+)0.2   1083 parent 9 4.4 ± 0.3 2.9 rDEN4-3702 NS2A 76.3 ± 0.3 0.2 rDEN4-7153 NS4B 4 5.9 ± 0.7 0.1 rDEN4-10634 3′ UTR 7 3.3 ±0.4 3.6 1136 parent 7 4.5 ± 0.4 1.2 rDEN4-3771 NS2A 4 6.4 ± 0.2 (+)0.1  rDEN4-4891 NS3 6 6.0 ± 0.5 0.3 rDEN4-10275 3′ UTR 4 6.7 ± 0.3 0.4 1189parent 13 2.3 ± 0.3 3.8 rDEN4-3303 NS1 4 6.3 ± 0.3 0.8 rDEN4-4812 NS3 56.1 ± 0.5 (+)0.5   rDEN4-5097 NS3 8 1.9 ± 0.1 4.3 rDEN4-7182 NS4B 6 6.3± 0.3 (+)0.7   ^(a)Reduction in mean virus titer (log₁₀PFU/ml) at 39° C.compared to permissive temperature (35° C.). ^(b)Groups of 6 sucklingmice were inoculated i.c. with 10⁴ PFU of virus. Brains were removed 5days later, homogenized, and titered in Vero cells. ^(c)Comparison ofmean virus titers of mice inoculated with mutant virus and concurrentDEN4 control. Bold denotes ≧50- or ≧100-fold decrease in replication insuckling or SCID-HuH-7 mice, respectively. ^(d)Groups of HuH-7-SCID micewere inoculated directly into the tumor with 10⁴ PFU virus. Serum wascollected on day 6 and 7 and titered in Vero cells. ^(e)Underlinedvalues indicate a 2.5 or 3.5 log₁₀PFU/ml reduction in titer in Verocells or HuH-7 cells, respectively, at indicated temp when compared topermissive temp (35° C.). ^(x)Small plaque size at 35° C.; small plaqueshave a diameter of <1.0 mm compared to wild type plaque diameter of1.5-2.0 mm in Vero cells, or a diameter of <0.4 mm compared to wild typeplaque diameter of 0.75 to 1.0 mm in HuH-7 cells.

TABLE 14 Phenotypes of rDEN4 mutant viruses encoding single mutationsidentified in 10 5-FU mutant viruses that are ts in both Vero and HuH-7cells. rDEN4- 5-FU Mutation Mean virus titer (log₁₀PFU/ml) at indicatedtemp (° C.) mutant (nt Gene/ Vero cells HuH-7 cells viruses position)region 35 37 39 39 Δ^(a) 35 37 38 39 Δ 239, parent 7.6 6.8 5.6  3.3 ^(e)4.3 7.6 6.7 4.7 2.5 5.1 489 773 4995^(f) NS3 5.7 4.9 3.6 <1.6  >4.1 6.45.7 4.0 <1.6  >4.8 473 parent 6.7 6.3 5.4 2.0 4.7 7.2 6.7 3.7 1.9 5.34480 NS2B 6.7 6.3 6.0 5.7 1.0 7.6 7.2 6.0 5.2 2.4 4995^(f) NS3 5.7 4.93.6 <1.6  >4.1 6.4 5.7 4.0 <1.6  >4.8 759 parent 7.2 6.9 6.4 4.7 2.5 7.56.8 6.3 3.1 4.4 4995^(f) NS3 5.7 4.9 3.6 <1.6  >4.1 6.4 5.7 4.0<1.6  >4.8 8020 NS5 7.1 6.6 6.7 5.9 1.2 7.4 7.1 6.1 5.4 2.0 816 parent6.8 6.4 5.8 3.9 2.9 7.5 6.2 5.5 3.1 4.4 4995^(f) NS3 5.7 4.9 3.6<1.6  >4.1 6.4 5.7 4.0 <1.6  >4.8 7174 NS4B 6.9 7.1 6.9 6.1 0.8 7.5 7.27.1 5.6 1.9 938 parent 7.1 6.5 5.6 3.1 4.0 7.2 6.4 5.6 3.1 4.1 3442 NS15.1 3.6 4.3 2.1 3.0 5.9 4.9 3.9 <1.6  4.3 4995^(f) NS3 5.7 4.9 3.6<1.6  >4.1 6.4 5.7 4.0 <1.6  >4.8 10275 3′UTR 6.9 6.4 6.4 5.8 1.1 7.16.8 7.1 5.2 1.9 173 parent 7.0 6.1 3.2 2.9 4.1 7.0 3.2 3.0 2.1 4.9 7163NS4B 7.8 7.7 7.6 7.2 0.6 8.0 7.7 7.5 7.4 0.6 7849 NS5 7.0 6.7 3.7 2.14.9 7.7 5.5 3.6 2.4 5.3 8872 NS5 7.0 6.3 6.4 4.4 2.6 7.4 6.4 5.1 2.9 4.5509 parent 6.2 5.8 5.5 3.4 2.8 6.5 6.1 4.5 <1.6  >4.9 4266 NS2B 5.9 6.16.1 5.2 0.7 6.7 6.1 5.7 5.3 1.4 8092 NS5  5.0^(x) 4.6 4.6 <1.6  >3.4 5.6^(x) 4.8 4.4 <1.6  >4.0 1033 parent 6.7 6.0 5.9 4.1 2.6 6.9 5.6 4.7<1.6  >5.3 4907 NS3 6.7 6.0 5.8 4.0 2.7 7.1 6.1 6.8 2.3 4.8 8730 NS5 7.06.7 6.6 6.7 0.3 7.6 7.0 7.2 6.6 1.0 9977 NS5 5.6 5.5 4.6 4.1 1.5 6.4 6.16.2 4.6 1.8 Replication in Replication in 7-day mice^(b) HuH-7-SCIDmice^(d) Mean log₁₀ Mean log₁₀ rDEN4- reduction reduction 5-FU Mutationfrom wt^(c) from wt^(c) mutant (nt (log₁₀PFU/g (log₁₀PFU/ml virusesposition) Gene/region n brain) n serum) 239, parent 30 2.1 6 0.3 489 7734995^(f) NS3 6 2.9 473 parent 12 1.2 8 (+)0.3   4480 NS2B 6 0.7 4995^(f)NS3 6 2.9 759 parent 12 1.4 5 (+)0.4   4995^(f) NS3 6 2.9 8020 NS5 6 0.5816 parent 6 2.9 6 0.4 4995^(f) NS3 6 2.9 7174 NS4B 6 0.6 938 parent 61.7 6 0.5 3442 NS1 6 4.1 4995^(f) NS3 6 2.9 10275 3′UTR 6 0.5 173 parent18 2.2 6 1.1 7163 NS4B 6 (+)0.1  7849 NS5 6 3.1 8872 NS5 6 0.1 509parent 6 1.9 6 1.5 4266 NS2B 6 1.0 8092 NS5 12 4.0 1033 parent 12 1.7 50.7 4907 NS3 12 1.8 8730 NS5 12 0.6 9977 NS5 6 0.7 ^(a)Reduction in meanvirus titer (log₁₀PFU/ml) at 39° C. compared to permissive temperature(35° C.). ^(b)Groups of 6 suckling mice were inoculated i.c. with 10⁴PFU of virus. Brains were removed 5 days later, homogenized, and titeredin Vero cells. ^(c)Comparison of mean virus titers of mice inoculatedwith mutant virus and concurrent DEN4 control. Bold denotes ≧50- or≧100-fold decrease in replication in suckling or SCID-HuH-7 mice,respectively. ^(d)Groups of HuH-7-SCID mice were inoculated directlyinto the tumor with 10⁴ PFU virus. Serum was collected on day 6 and 7and titered in Vero cells. ^(e)Underlined values indicate a 2.5 or 3.5log₁₀PFU/ml reduction in titer in Vero cells or HuH-7 cells,respectively, at indicated temp when compared to permissive temp (35°C.). ^(f)Data represents the results from a single rDEN4-4995 virus.^(x)Small plaque size at 35° C.; small plaques have a diameter of <1.0mm compared to wild type plaque diameter of 1.5-2.0 mm in Vero cells, ora diameter of <0.4 mm compared to wild type plaque diameter of 0.75 to1.0 mm in HuH-7 cells.

TABLE 15 sp, ts and mouse attenuation phenotypes of rDEN4 mutant virusesencoding single mutations identified in 3 HuH-7 cell-specific ts 5-FUmutant viruses. Replication in 7-day mice^(b) Replication in HuH-7-SCIDrDEN4- Mean log₁₀ mice^(b) 5-FU Mutation Mean virus titer (log₁₀PFU/ml)at indicated temp (° C.) reduction from Mean log₁₀ mutant (nt Gene/ Verocells HuH-7 cells wt^(c)(log₁₀PFU/g reduction from wt^(c) virusesposition) region 35 37 39 39 Δ^(a) 35 37 38 39 Δ n brain)₁₀ n(log₁₀PFU/ml serum) 686 parent 7.0 6.7 6.7 6.4 0.6 7.3 6.8 6.4 2.2 5.112 3.8 6 1.2 3575 NS2A 6.9 6.9 7.1 7.0 0.1 7.9 6.8 6.9 4.9 3.0 12 2.3 nd^(e) 4062 NS2A 6.8 6.6 6.3 4.7 2.1 6.9 6.8 7.0 <1.6  >5.3 12 2.2 nd7163 NS4B 7.8 7.7 7.6 7.2 0.6 8.0 7.7 7.5 7.4 0.6 6 (+)0.1  nd 992parent 7.3 7.1 6.8 5.9 1.4 7.4 6.9 5.0 <1.6  >5.8 6 2.7 7 1.3 5695 NS35.6 4.7 4.7 3.8 1.8 6.3 5.1 3.7 <1.6  >4.7 6 2.8 nd 7162 NS4B 7.2 7.36.6 6.8 0.4 7.4 7.3 7.3 6.6 0.8 8 0.3 nd 1175 parent 7.4 7.1 6.9 5.3 2.17.6 6.5 4.7 3.3 4.3 12 1.7 5 1.0 7153 NS4B 7.7 7.7 7.6 7.2 0.5 8.0 7.87.5 6.9 1.1 6 0.2 nd 10186 NS5 4.3 3.7 2.4 <1.6  >2.7 5.1 <1.6  <1.6 <1.6  >3.5 6 3.4 nd 10275 3′ UTR 6.9 6.4 6.4 5.8 1.1 7.1 6.8 7.1 5.2 1.96 0.5 nd ^(a)Reduction in titer (log₁₀PFU/ml) at 39° C. compared topermissive temperature (35° C.). ^(b)Groups of 6 suckling mice wereinoculated i.c. with 10⁴ PFU virus. Brains were removed 5 days later,homogenized, and titered in Vero cells. ^(c)Determined by comparing meanviral titers of mice inoculated with mutant virus and concurrent 2A-13or rDEN4 wt control. ^(d)Underlined values indicate a 2.5 or 3.5log₁₀PFU/ml reduction in titer in Vero cells or HuH-7 cells,respectively, at indicated temp when compared to permissive temp (35°C.).

TABLE 16 Temperature-sensitive (ts) and mouse brain attenuation (att)phenotypes of additional rDEN4 viruses encoding single 5-FU mutations.Gene/ 5-FU region Mean virus titer (log₁₀PFU/ml) at indicated temp (°C.) mutant containing Vero cells HuH-7 cells virus Virus mutation 35 3738 39 Δ^(a) 35 37 38 39 Δ 695 rDEN4-40 5′ UTR 7.4 7.2 6.7 6.2 1.2 7.67.5 7.1 5.8 1.8 718 rDEN4-4059 NS2A 7.0 6.7 6.4 6.2 0.8 7.7 7.1 7.0 6.61.1 311 rDEN4-4896 NS3 7.0 6.1 5.9 4.2 2.8  6.9^(x) 6.0 5.6 3.3 3.6 695rDEN4-6106 NS3 6.8 6.3 5.9 3.9 2.9 7.1 6.0 5.2 3.4 3.7 631 rDEN4-6259NS3 7.0 6.1 5.8 5.0 2.0 7.5 6.6 5.7 4.2 3.3 695^(e) rDEN4-7546 NS4B 7.57.6 7.4 6.6 0.9 7.7 7.6 7.3 5.7 2.0 718 rDEN4-7630 NS5 7.0 6.9 6.9 6.40.6 7.4 7.4 7.2 6.8 0.6 718 rDEN4-8281 NS5 6.4 6.6 6.7 5.4 1.0 7.6 7.67.0 5.1 2.5 Replication in suckling mice^(b) Gene/ Mean virus 5-FUregion titer ± SE Mean log₁₀-unit mutant containing (log₁₀PFU/greduction from virus Virus mutation n brain) value for wt^(c) 695rDEN4-40 5′ UTR  nd^(f) nd 718 rDEN4-4059 NS2A nd nd 311 rDEN4-4896 NS36 4.1 ± 0.4 2.0** 695 rDEN4-6106 NS3 nd nd 631 rDEN4-6259 NS3 6 2.2 ±0.2 3.9** 695^(e) rDEN4-7546 NS4B nd nd 718 rDEN4-7630 NS5 6 5.0 ± 0.30.5  718 rDEN4-8281 NS5 6 5.0 ± 0.5 1.1  ^(a)Reduction in titer(log₁₀PFU/ml) at 39° C. compared to titer at permissive temperature (35°C.). ^(b)6 mice were inoculated i.c. with 10⁴ PFU virus in 30 μlinoculum. Brains were removed 5 days later, homogenized, and titered onVero cells. Limit of detection is 2.0 log₁₀PFU/g. ^(c)Determined bycomparing mean viral titers of mice inoculated with sample virus and wtrDEN4 control. ^(d)Underlined values indicate a 2.5 or 3.5 log₁₀PFU/mlreduction in titer in Vero cells or HuH-7 cells, respectively, atindicated temperature when compared to permissive temperature (35° C.).^(e)The 7546 mutation is also present in nine other 5-FU mutant viruses.^(x)Small plaque size at 35° C.; small plaques have a diameter of <0.4mm compared to wt plaque diameter of 0.75 to 1.0 mm in HuH-7 cells.^(f)not determined **The att phenotype is defined as a reduction of >1.5log₁₀PFU/g compared to wt virus.

TABLE 17 Growth of wt DEN-4 2A-13 in SCID mice transplanted with HuH-7cells.^(a) Dose Virus titer (log₁₀PFU/ log₁₀PFU/ml serum log₁₀PFU/gtissue ml) Mouse # day 3 day 5 Brain Liver Tumor 4 87 2.7 5.9 2.0 6.98.0 88 2.0 5.9 3.8 3.3 8.0 89 <1.7 6.2 2.7 3.6 8.0 90 1.7 3.5 3.2 3.07.0 5 84 <1.7 7.2 3.2 4.0 7.0 85 1.7 6.6 3.6 6.3 5.8 6 91 4.4 8.3 6.07.3 8.0 92 4.2 7.7 3.3 6.9 7.3 93 4.0 6.6 3.3 5.7 8.4 94 4.3 8.1 5.8 7.87.5 ^(a)SCID mice were injected i.p. with 10⁷HuH-7 human hepatoma cells.Approximately 8 weeks later, groups of tumor-bearing SCID-HuH-7 micewere inoculated with virus directly into the tumor. Serum and tissueswere collected on day 5, processed, and titered in Vero cells.

TABLE 18 Combination of ts mutations, NS3 4995 and NS5 7849, in rDEN4results in an additive ts phenotype. Replication in suckling mice^(b)Mean virus titer (log₁₀PFU/ml) Mean virus at indicated temp (° C.) titer± SE Mean log₁₀ Vero cells HuH-7 cells (log₁₀PFU/g reduction Virus 35 3738 39 Δ^(a) 35 37 38 39 Δ brain) from wt^(c) 2A-13 wt 7.1 7.1 6.9 6.80.3 7.4 7.3 6.7 6.4 1.0 6.9 ± 0.09 — rDEN4 wt 7.0 6.8 6.6 6.4 0.6 7.57.3 6.7 6.4 1.1 6.5 ± 0.11 — rDEN4Δ30 7.0 6.7 6.2 6.2 0.8 7.5 7.0 6.55.1 2.4 5.9 ± 0.21 0.6 rDEN4-4995 5.7 4.9 3.6 <1.6 ^(d ) >4.1 6.4 5.74.0 <1.6  >4.8 3.4 ± 0.10 3.1 rDEN4-7849 7.0 6.7 3.7 2.1 4.9 7.7 5.5 3.6 2.4  5.3 2.6 ± 0.29 3.9 rDEN4-4995-7849 5.9 2.8 <1.6  <1.6  >4.3 5.62.4 <1.6  <1.6  >4.0 2.3 ± 0.20 4.2 ^(a)Reduction in titer (log₁₀PFU/ml)at 39° C. compared to titer at permissive temperature (35° C.).^(b)Groups of 6 suckling mice were inoculated i.c. with 10⁴ PFU virus.Brains were removed 5 days later, homogenized, and titered in Verocells. The limit of detection is 2.0 log₁₀PFU/g. ^(c)Determined bycomparing mean viral titers of mice inoculated with sample virus andrDEN4 wt control. ^(d)Underlined values indicate a 2.5 or 3.5log₁₀PFU/ml reduction in titer in Vero cells or HuH-7 cells,respectively, at indicated temperature when compared to permissivetemperature.

TABLE 19 The 5-FU mutations are compatible with the Δ30 mutation forreplication in the brain of suckling mice. Mean virus Mean No. of titer± log₁₀-unit mice/ SE (log₁₀PFU/g reduction Virus group brain)^(a) fromwt^(b) rDEN4 12 6.0 ± 0.1 — rDEN4Δ30 12 5.3 ± 0.1 0.7 rDEN4-2650^(c) 123.7 ± 0.2 2.3 rDEN4Δ30-2650 12 3.9 ± 0.1 2.1 rDEN4-4995^(d) 6 3.5 ± 0.22.5 rDEN4Δ30-4995 6 2.7 ± 0.4 3.3 rDEN4-8092^(d) 12 2.0 ± 0.1 4.0rDEN4Δ30-8092 6 3.2 ± 0.2 2.8 rDEN4-10634^(c) 12 3.8 ± 0.1 2.2rDEN4Δ30-10634 12 3.6 ± 0.1 2.4 ^(a)Groups of 6 suckling mice wereinoculated i.c. with 10⁴ PFU of virus. Brains were removed 5 days later,homogenized, and titered in Vero cells. ^(b)Comparison of mean virustiters of mice inoculated with mutant virus and rDEN4 control.^(c)Mutation restricts growth in both mouse brain and HuH-7-SCID mice.^(d)Mutation restricts growth in mouse brain only. The 8092 mutation hasnot been tested in SCID-HuH7 mice.

TABLE 20 Temperature-sensitive and mouse brain attenuation phenotypes ofviruses bearing charge-cluster-to-alanine mutations in the NS5 gene ofDEN4. Mean virus liter (log₁₀PFU/ml at indicated temperature (° C.)^(b)Changed # nt Vero Cells HuH-7 Cells Mutation^(a) AAPair changed 35 37 3839 Δ^(c) 35 37 38 39 Δ wt (rDEN4) n/a 0 8.1 8.1 7.9 7.6 0.5 8.3 8.0 7.5 7.5 0.8 deletion n/a 30 6.3 6.1 6.1 5.7 0.6 6.9 6.3 5.9  4.7 2.2(rDEN4Δ30) 21-22 D R 4 7.2 6.8 6.7 6.1 1.1 7.6 7.1 7.0  4.7 2.9 22-23 RK 4 7.0 7.8 6.9 3.7 3.3 7.6 7.6 6.5 <1.7 >5.9 23-24 K E 3 6.7 6.6 6.06.5 0.2 7.1 7.3 5.6 <1.7 >5.4 26-27 E E 3 7.8 7.6 6.8 4.0 3.8 8.4 8.27.3  4.9 3.5 46-47 K D 3 7.4 7.4 7.3 7.0 0.4 7.8 7.8 7.3  6.8 1.0157-158 E E 3 6.5 7.2 5.1 5.1 1.4 7.6 7.4 5.9 <1.7 >5.9 200-201 K H 45.3 4.6 5.3 4.1 1.2 5.6 4.9 3.7 <1.7 >3.9 246-247 R H 5 6.9 5.8 5.7 5.41.5 6.4 6.1 6.1  5.5 0.9 253-254 E K 4 7.1 6.9 6.8 7.0 0.1 7.9 7.5 7.6 6.8 1.1 356-357 K E 3 7.7 7.6 7.0 7.0 0.7 8.0 7.3 6.4 <1.7 >6.3 387-388K K 5 7.7 6.1 7.0 <1.7  >6.0 7.0 6.3 7.0 <1.7 >5.3 388-389 K K 5 5.1 4.5<1.7  <1.7  >3.4 6.1 5.0 <1.7  <1.7 >4.4 396-397 R E 4 7.0 7.3 6.5 5.51.5 7.5 7.6 7.5 <1.7 >5.8 397-398 E E 2 7.0 7.1 7.0 3.0 4.0 8.0 7.6 7.0<1.7 >6.3 436-437 D K 4 4.5 3.3 3.0 2.0 2.5 5.7 4.5 <1.7  <1.7 >4.0500-501 R E 3 6.6 6.3 5.7 2.3 4.3 7.1 6.5 <1.7  <1.7 >5.4 520-521 E E 35.6 4.7 4.3 <1.7  >3.9 6.7 5.7 <1.7  <1.7 >5.0 523-524 D K 4 6.6 6.3 6.35.8 0.8 7.1 6.6 <1.7  <1.7 >5.4 524-525 K K 5 7.1 6.9 6.9 6.6 0.5 7.87.4 7.0  5.3 2.5 525-526 K D 4 7.8 7.1 7.6 6.8 1.0 7.9 7.7 8.0  6.9 1.0596-597 K D 3 4.6 4.0 2.6 <1.7  >2.9 5.7 4.9 4.0 <1.7 >4.0 641-642 K E 47.3 6.9 6.9 5.2 2.1 7.8 7.5 7.2  6.9 0.9 642-643 E R 3 6.8 6.1 4.0 3.33.5 7.5 7.1 6.6  3.0 4.5 645-646 E K 4 6.3 5.3 5.9 3.1 3.2 6.4 5.8 5.5 4.5 1.9 649-650 K E 3 6.9 6.8 6.9 6.3 0.6 7.1 7.3 7.5  7.0 0.1 654-655D R 4 6.3 5.7 <1.7  <1.7  >4.6 7.0 7.1 4.6 <1.7 >5.3 750-751 R E 3 7.17.1 6.9 5.7 1.4 7.8 6.9 6.5  5.6 2.2 808-809 E D 3 4.6 4.1 <1.7 <1.7  >2.9 5.2 <1.7  <1.7  <1.7 >3.5 820-821 E D 2 6.3 6.3 5.6<1.7  >4.6 6.9 6.0 5.7 <1.7 >5.2 827-828 D K 4 6.9 6.3 6.3 5.9 1.0 7.56.9 5.0 <1.7 >5.8 877-878 K E 3 7.6 7.3 7.0 7.0 0.6 7.9 7.9 7.3  5.8 2.1878-879 E E 3 7.6 7.3 7.3 7.1 0.5 8.1 8.1 7.9  6.6 1.5 Replication insuckling mice^(d) Mean titer ± SE Changed # nt (log₁₀PFU/g Mean logMutation^(a) AA Pair changed n brain) reduction from wt^(c) wt (rDEN4)n/a 0 48 6.0 ± 0.16 — deletion n/a 30 42 5.4 ± 0.22 0.6 (rDEN4Δ30) 21-22D R 4 6 5.0 ± 0.50 0.6 22-23 R K 4 6 2.6 ± 0.19 2.9 23-24 K E 3 18 4.7 ±0.09 1.5 26-27 E E 3 6 5.7 ± 0.30 +0.1 46-47 K D 3 6 5.4 ± 0.42 0.5157-158 E E 3 6 2.8 ± 0.31 2.7 200-201 K H 4 12 5.5 ± 0.45 0.8 246-247 RH 5 6 6.1 ± 0.17 +0.5 253-254 E K 4 6 6.2 ± 0.13 +0.6 356-357 K E 3 63.5 ± 0.58 2.0 387-388 K K 5 6 3.1 ± 0.33 2.4 388-389 K K 5 6 5.0 ± 0.231.4 396-397 R E 4 18 5.4 ± 0.35 1.1 397-398 E E 2 6 6.0 ± 0.22 0.8436-437 D K 4 12 2.3 ± 0.14 3.9 500-501 R E 3 6 6.9 ± 0.49 +0.7 520-521E E 3 6 5.2 ± 0.48 0.2 523-524 D K 4 6 4.2 ± 0.47 1.3 524-525 K K 5 63.4 ± 0.54 2.1 525-526 K D 4 6 3.7 ± 0.64 1.8 596-597 K D 3 6 5.9 ± 0.140.5 641-642 K E 4 6 4.7 ± 0.45 1.2 642-643 E R 3 12 2.6 ± 0.15 3.6645-646 E K 4 6 5.4 ± 0.51 0.2 649-650 K E 3 12 6.4 ± 0.20 +0.2 654-655D R 4 12 1.8 ± 0.10 4.0 750-751 R E 3 6 6.0 ± 0.18 0.7 808-809 E D 3 61.8 ± 0.05 3.1 820-821 E D 2 6 5n5 ± 0.33 1.2 827-828 D K 4 6 3.6 ± 0.762.3 877-878 K E 3 12 4.4 ± 0.65 1.8 878-879 E E 3 12 2.4 ± 0.10 3.8^(a)Positions of the amino acid pair mutated to an alanine pair;numbering starts at the amino terminus of the NS5 protein.^(b)Underlined values indicate a 2.5 or 3.5 log10 PFU/ml reduction intiter in Vero or HuH-7 cells, respectively, at the indicatedtemperatures when compared to permissive temperature (35° C.).^(c)Reduction in titer (log10 PFU/ml) at 39° C. compared to permissivetemperature (35° C.). ^(d)Groups of six mice were inoculated i.c. with4.0 log10 PFU virus in a 30 μl inoculum. The brain was removed 5 dayslater, homogenized, and titered in Vero cells. ^(e)Determined bycomparing mean viral titers in mice inoculated with sample virus andconcurrent wt controls (n = 6). The attenuation phenotype is defined asa reduction of ≧1.5 log10 PFU/g compared to wt virus; reductions of ≧1.5are listed in boldface.

TABLE 21 SCID-HuH-7 attenuation phenotypes of viruses bearing charge-cluster-to-alanine mutations in the NS5 gene of DEN4. Replication inSCID-HuH-7 mice^(b) Mean peak virus Mean log AA titer ± SE reductionMutation^(a) changed n (log₁₀PFU/ml serum) from wt^(c) wt na 21 5.4 ±0.4 — Δ30 na 4 3.7 ± 0.6 2.5 23-24 KE 19 4.7 ± 0.5 1.3 157-158 EE 6 4.6± 0.6 1.3 200-201 KH 12 3.7 ± 0.2 2.6 356-357 KE 10 6.3 ± 0.7 (−) 1.1  396-397 RE 12 4.4 ± 1.3 1.2 397-398 EE 6 6.0 ± 0.5 (−) 0.1   436-437 DK6 3.6 ± 0.2 2.6 500-501 RE 8 5.1 ± 0.4 1.1 523-524 DK 5 5.3 ± 0.7 0.6750-751 RE 8 5.1 ± 0.4 1.1 808-809 ED 8 3.2 ± 0.4 3.0 827-828 DK 5 2.9 ±0.2 1.6 878-879 EE 5 4.4 ± 0.7 1.5 ^(a)Positions of the amino acid pairchanged to a pair of alanines; numbering starts at the amino terminus ofthe NS5 protein. ^(b)Groups of SCID-HuH-7 mice were inoculated directlyinto the tumor with 10⁴ PFU virus. Serum was collected on days 6 and 7and titered in Vero cells. ^(c)Comparison of mean virus titers of miceinoculated with mutant virus and concurrent DEN4 control. Bold denotes a≧100-fold decrease in replication. A (−) sign indicates an increase inreplication relative to wt.

TABLE 22 Combination of paired charge-cluster-to-alanine mutations intodouble-pair mutant viruses. Mutation Pair 1 Mutation Pair 2 Recovered23-24 200-201 Yes 23-24 356-357 Yes 23-24 396-397 Yes 23-24 523-524 Yes23-24 827-828 No 157-158 200-201 No 157-158 356-357 No 157-158 396-397No 157-158 523-524 Yes 157-158 827-828 No 827-828 200-201 No 827-828356-357 No 827-828 396-397 Yes 827-828 523-524 No

TABLE 23 Temperature-sensitive and mouse brain attenuation phenotypes ofdouble charge-cluster-to-alanine mutants of the NS5 gene of rDEN4. Meanvirus titer (log10 PFU/ml) at indicated temperature (° C.)^(b) Charged#nt Vero Cells HuH-7 cells Mutation^(a) AA Pair changed 35 37 38 39Δ^(c) 35 37 38 39 Δ wt n/a 0 8.1 8.1 7.9 7.6 0.5 8.3 8.0 7.5  7.5 0.8Δ30 n/a 30 6.3 6.1 6.1 5.7 0.6 6.9 6.3 5.9  4.7 2.2 23-24 K E 3 6.7 6.66.0 6.5 0.2 7.1 7.3 5.6 <1.7 >5.4 200-201 K H 4 5.3 4.6 5.3 4.1 1.2 5.64.9 3.7 <1.7 >3.9 23-24; 200-201 K E, K H 7 7.1 6.5 6.6 <1.7  >5.4 7.87.3 <1.7  <1.7 >6.1 23-24 K E 3 6.7 6.6 6.0 6.5 0.2 7.1 7.3 5.6<1.7 >5.4 356-357 K E 3 7.7 7.6 7.0 7.0 0.7 8.0 7.3 6.4 <1.7 >6.3 23-24;356-357 K E, K E 6 23-24 K E 3 6.7 6.6 6.0 6.5 0.2 7.1 7.3 5.6 <1.7 >5.4396-397 R E 4 7.0 7.3 6.5 5.5 1.5 7.5 7.6 7.5 <1.7 >5.8 23-24; 396-397 KE, R E 7 6.3 4.9 <1.7  <1.7  >4.6 7.1 6.0 5.6 <1.7 >5.4 157-158 E E 36.5 7.2 5.1 5.1 1.4 7.6 7.4 5.9 <1.7 >5.9 396-397 R E 4 7.0 7.3 6.5 5.51.5 7.5 7.6 7.5 <1.7 >5.8 157-158; 396-397 E E, R E 7 157-158 E E 3 6.57.2 5.1 5.1 1.4 7.6 7.4 5.9 <1.7 >5.9 523-524 D K 4 6.6 6.3 6.3 5.8 0.87.1 6.6 <1.7  <1.7 >5.4 157-158; 523-524 E E, D K 7 5.6 3.9 <1.7 <1.7  >3.9 6.3 4.1 <1.7  <1.7 >4.6 396-397 R E 4 7.0 7.3 6.5 5.5 1.5 7.57.6 7.5 <1.7 >5.8 827-828 D K 4 6.9 6.3 6.3 5.9 1.0 7.5 6.9 5.0<1.7 >5.8 396-397; 827-828 R E, D K 8 7.0 6.5 6.0 <1.7  5.3 >6.7 5.7<1.7  <1.7 >5.0 Replication in suckling mice^(d) Mean virus titer ± SEMean log (log₁₀PFU/g reduction Mutation^(a) Charged AA Pair #nt changedn brain) from wt^(e) wt n/a 0 48 6.0 ± 0.16 — Δ30 n/a 30 42 5.4 ± 0.220.6 23-24 K E 3 18 4.7 ± 0.09 1.5 200-201 K H 4 12 5.5 ± 0.45 0.8 23-24;200-201 K E, K H 7 6 5.8 ± 0.16 0.6 23-24 K E 3 18 4.7 ± 0.09 1.5356-357 K E 3 6 3.5 ± 0.58 2.0 23-24; 356-357 K E, K E 6 23-24 K E 3 184.7 ± 0.09 1.5 396-397 R E 4 18 5.4 ± 0.35 1.1 23-24; 396-397 K E, R E 76 3.7 ± 0.44 2.7 157-158 E E 3 6 2.8 ± 0.31 2.7 396-397 R E 4 18 5.4 ±0.35 1.1 157-158; 396-397 E E, R E 7 6 2.0 ± 0.12 4.8 157-158 E E 3 62.8 ± 0.31 2.7 523-524 D K 4 6 4.2 ± 0.47 1.3 157-158; 523-524 E E, D K7 396-397 R E 4 6 4.8 ± 0.54 1.6 827-828 D K 4 6 3.6 ± 0.76 2.3 396-397;827-828 R E, D K 8 6 4.7 ± 0.10 1.2 ^(a)Positions of the amino acid pairmutated to an alanine pair; numbering starts at the amino terminus ofthe NS5 protein. ^(b)Underlined values indicate a 2.5 or 3.5 log₁₀PFU/ml reduction in titer in Vero or HuH-7 cells respectively, at theindicated temperatures when compared to permissive temperature (35° C.).^(c)Reduction in titer (log₁₀PFU/ml) at 39° C. compared to permissivetemperature (35° C.). ^(d)Groups of six suckling mice were inoculatedi.c. with 4.0 log₁₀PFU virus in a 30 μl inoculum. Brains were removed 5days later, homogenized, and titered in Vero cells. ^(e)Determined bycomparing mean viral titers in mice inoculated with sample virus andconcurrent wt controls (n = 6); reductions ≧1.5 are listed in boldface.

TABLE 24 SCID-HuH-7 attenuation phenotypes of double charge-cluster-to-alanine mutants of the NS5 gene of rDEN4. Replication inSCID-HuH-7 mice^(b) Mean peak virus Mean log Charged titer ± SEreduction Mutation^(a) AA Pair n (log₁₀PFU/ml serum) from wt^(c) wt n/a21 5.4 ± 0.4 —   Δ30 n/a 4 3.7 ± 0.6 2.5 23-24 K E 19 4.7 ± 0.5 1.3200-201 K H 12 3.7 ± 0.2 2.6  23-24; 200-201 K E, K H 13 3.4 ± 0.1 2.923-24 K E 19 4.7 ± 0.5 1.3 356-357 K E 10 6.3 ± 0.7 (+) 1.1    23-24;356-357 K E, K E 4 3.6 ± 0.3 2.3 23-24 K E 19 4.7 ± 0.5 1.3 396-397 R E12 4.4 ± 1.3 1.2  23-24; 396-397 K E, R E 10 3.4 ± 0.5 3.3 157-158 E E 64.6 ± 0.6 1.3 396-397 R E 12 4.4 ± 1.3 1.2 157-158; 396-397 E E, R E 62.2 ± 0.2 3.6 157-158 E E 6 4.6 ± 0.6 1.3 523-524 D K 5 5.3 ± 0.7 0.6157-158; 523-524 E E, D K 3 5.1 ± 0.6 0.8 396-397 R E 12 4.4 ± 1.3 1.2827-828 D K 5 2.9 ± 0.2 1.6 396-397; 827-828 R E, D K 4 4.1 ± 0.7 0.4^(a)Positions of the amino acid pair mutated to an alanine pair;numbering starts at the amino terminus of the NS5 protein. ^(b)Groups ofSCID-HuH-7 mice were inoculated directly into the tumor with 10⁴ PFU ofvirus. Serum was collected on days 6 and 7 and titered in Vero cells.^(c)Comparison of mean virus titers of mice inoculated with mutant virusand concurrent DEN4 control. Bold denotes a ≧100-fold decrease inreplication. A (+) sign indicates an increase in replication relative towt.

TABLE 25 Phenotypes (temperature sensitivity, plaque size andreplication in mouse brain and SCID-HuH-7 mice) of wt DEN4 and virusescontaining the Δ30 and 7129 mutations. Replication in suckling mouseReplication in SCID-HuH-7 brain^(c) mice^(e) Mean virus titer (log₁₀PFU/ml) at Mean virus Mean peak virus indicated temperature (° C.) titer± SE Mean log titer ± SE Mean log VERO HUH7 C6/36 (log₁₀PFU/g reduction(log₁₀PFU/ml reduction Virus ID Mutation^(a) 35 39 Δ^(b) 35 39 Δ 32 nbrain) from wt^(d) n serum)^(f) from wt^(d) 1-TD-1A wt 7.3 6.8 0.5 8 6.81.2 8.3 36 6.1 ± 0.21 — 21 5.4 ± 0.4 — p4Δ30 Δ30 6.6 6.5 0.1 7.4 6.4 1.042 5.4 ± 0.22 0.6 4 3.7 ± 0.6 2.5 5-1A1 C7129U 6.7 6.5 0.2 7.5 6 1.57.6* 6 6.2 ± 0.30 0.0 rDEN4-7129-1A C7129U 7.3 7.0 0.3 7.6 6.3 1.3 7.5*6 7.2 ± 0.12 (−) 0.4 4 5.4 ± 0.8 (−) 0.8 rDEN4Δ30-7129 C7129U + Δ30 7.07.1* ^(a)Position and identity of the mutated nucleotides. ^(b)Reductionin titer (log₁₀ PFU/ml) at 39° C. compared to permissive temperature(35° C.). ^(c)Groups of six suckling mice were inoculated i.c. with 4.0log₁₀ PFU virus in a 30 μl inoculum. The brain was removed 5 days later,homogenized, and titered in Vero cells. ^(d)Determined by comparing meanviral titers in mice inoculated with sample virus and concurrent wtcontrols (n = 6). The attenuation phenotype is defined as a ≧50- or≧100-fold decrease in replication in suckling or SCID-HuH-7 mice,respectively. A (−) sign indicates an increase in replication relativeto the wt control. ^(e)Groups of SCID-HuH-7 mice were inoculateddirectly into the tumor with 10⁴ PFU virus. Serum was collected on days6 and 7 and titered in Vero cells. *Small plaque size.

TABLE 26 The 5-fluorouracil 5-1A1 small plaque mutant demonstrates arestriction of midgut infection following oral infection of Aedesaegytpi mosquitoes. Dose No. Virus ingested mosquitoes Midgut-onlyDisseminated Total no. tested (log₁₀PFU) ^(a) tested infection ^(b)infection ^(c) infected ^(d, e) wtDEN4 4.5 19 1 (5%)  17 (89%) 18 (95%)(2A-13) 3.5 26 9 (35%)  7 (27%) 16 (62%) 2.5 28 1 (4%)  0 1 (4%) OID₅₀ =3.9 OID₅₀ = 3.3 5-1A1 3.5 34 4 (12%) 2 (6%)  6 (18%) 2.5 9 0  1 (11%)  1(11%) 1.5 23 0 0 0 OID₅₀ ≧ 3.9 ^(a) Amount of virus ingested, assuming a2 μl bloodmeal. ^(b) Number (percentage) of mosquitoes with detectabledengue virus antigen in midgut tissue, but no detectable dengue virusantigen in head; mosquitoes were assayed 21 days post-feed, and denguevirus antigen was identified by IFA. ^(c) Number (percentage) ofmosquitoes with detectable dengue virus antigen in both midgut and headtissue. ^(d) Total number (percentage) of mosquitoes with detectabledengue virus antigen. ^(e) The proportion of total infections caused bywild type DEN4 was significantly higher than the proportion caused by5-1A1 (logistic regression, N = 426, P < 0.0001). There were too fewdisseminated infection caused by 5-1A1 to permit statistical analysis.

TABLE 27 The 5-fluorouracil 5-1A1 small plaque mutant demonstrates arestriction of infection following intrathoracic inoculation ofToxorhynchites splendens mosquitoes. Dose No. Virus ingested mosquitoesNo (%) tested (log₁₀PFU) ^(a) tested infected ^(c) wtDEN4 4.0 5 5 (100)(2A-13) 3.0 4 4 (100) 2.0 4 1 (25)  MID₅₀ = 2.3 log₁₀PFU 5-1A1 3.0 9 02.0 7 1 (14)  1.0 7 0 MID₅₀ > 3.0 log₁₀PFU ^(a) Amount of virusinoculated in a 0.22 μl inoculum. ^(b) Number (percentage) of mosquitoeswith detectable dengue virus antigen in head tissue; mosquitoes wereassayed 14 days post-inoculation, and dengue virus antigen wasidentified by IFA. ^(c) The proportion of infections caused by wild typeDEN4 was significantly higher than the proportion caused by 5-1A1(logistic reression, N = 36, P < 0.01).

TABLE 28Mutagenesis primers for the deletion or swap of sequences in DEN4 showingconserved differences from tick-borne flaviviruses. DEN4 Type of SEQnucleotides¹ mutation² Mutagenesis Primer³ ID NO 10508-10530 ΔCTGGTGGAAGCCCAACACAAAAAC 64 10508-10530 swapCTGGTGGAAGGAAGAGAGAAATTGGCAACTCCCCAACACAAAAAC 65 10535-10544 ΔAGACCCCCCCAAGCATATTGAC 66 10535-10544 swapAGACCCCCCCAATATTTCCTCCTCCTATAGCATATTGAC 67 10541-10544 ΔCCCAACACAAAGCATATTGAC 68 ¹Nucleotides numbered 5′ to 3′, in the oppositedirection from FIG. 5.3 ²AΔ: deletion of specified DEN4 nucleotides;swap: exhange of specified DEN4 nucleotides with homologous sequencefrom Langat ³no swap mutation was made for nucleotides 10541-10544

TABLE 29 Virus titer and plaque size of 3′ UTR mutant viruses in Veroand C6/36 cells. Vero C6/36 Titer Titer (log₁₀ Plaque (log₁₀ PlaqueVirus PFU/ml) size¹ PFU/ml) size rDEN4Δ10508-10530 8.1 wt 7.5 wtrDEN4swap10508-10530 5.4 sp 6.6 wt rDEN4Δ10535-10544 5.8 wt 7.0 sprDEN4swap10535-10544 7.0 wt 7.3 wt rDEN4Δ10541-10544 6.4 wt >7.0 wt¹Plaque size is designated as equivalent to wild type (wt) or ≦50% ofwild type (sp) on the designated cell type.

TABLE 30 Infectivity of wt DEN4 and 3′ UTR mutants for Toxorhynchitessplendens via intrathoracic inoculation. No. Dose mosquitoes % MID₅₀Virus (log₁₀PFU)^(a) tested Infected^(b) (log₁₀ PFU) rDEN4 wt 3.3 6 832.3 2.3 7 57 1.3 6 0 0.3 6 0 rDEN4Δ10508- 4.4 8 0 10530 3.4 9 11 2.4 4 0^(a)Amount of virus inoculated in a 0.22 μl inoculum. ^(b)Percentage ofmosquitoes with detectable dengue virus antigen in head tissue;mosquitoes were assayed 14 days post-inoculation, and dengue virusantigen was identified by IFA

TABLE 31 Infectivity of 3′ UTR swap mutant viruses for Aedes aegypti fedon an infectious bloodmeal. Dose No. Mos- Virus ingested quitoes TotalNo. Disseminated Tested (log₁₀PFU)^(a) Tested Infected^(b,c)Infections^(c,d) rDEN4 3.8 18 11 (61%)   4 (22%) 2.8 15 5 (33%) 1 (6%)1.8 15 0 0 OID₅₀ = 3.4  OID₅₀ = ≧4.2 rDEN4swap 3.8 25 5 (20%) 2 (8%)10535-10544 2.8 25 0 0 1.8 20 0 0 OID₅₀ = ≧4.2 ^(a)Amount of virusingested, assuming a 2 μl bloodmeal. ^(b)Number (%) of mosquitoes withdetectable dengue virus antigen in the midgut tissue; mosquitoes wereassayed either 14 d post-feed and dengue virus antigen was identified byIFA. ^(c)At a dose of 3.8 log₁₀PFU, rDEN4swap10535-10544 infectedsignificantly fewer mosquitoes at the midgut than wt rDEN4 (Fisher'sexact test, N = 43, P < 0.01), although disseminated infections were notsignificantly different (Fisher's exact test, N = 43, P = 0.38).^(d)Number (%) of mosquitoes with detectable dengue virus antigen in thehead tissue.

TABLE 32 Putative Vero cell adaptation mutations derived from the set of5-FU mutant viruses and other DEN4 viruses passaged in Vero cells. OtherDEN viruses passaged 5-FU mutant viruses in Vero cells NucleotideGene/region Nucleotide Amino acid No. of viruses Nucleotide Amino acidposition (a.a. #)^(b) change change with the mutation Virus changechange  1455 E (452) G > U val > phe 5  2280^(1,2,3) E (727) U > C phe >leu 2  4891^(2,3) NS3 (1597) U > C ile > thr 2  4995^(1,2) NS3 (1599)U > C ser > pro 8  7153 NS4B (2351) U > C val > ala 3 2AΔ30 U > C val >ala  7162 NS4B (2354) U > C leu > ser 4 2A-1 U > C leu > ser  7163 NS4B(2354) A > U or C leu > phe 7 rDEN4Δ30 A > U leu > phe 2A-13-1A1 A > Uleu > phe  7182^(1,2,3) NS4B (2361) G > A gly > ser 2  7546 NS4B (2482)C > U ala > val 10  7630³ NS5 (2510) A > G lys > arg 1 814669 A > Glys > arg 10275 3′ UTR A > U n/a^(c) 6 10279 3′ UTR A > C n/a 4 ^(a)Conservation with DEN1, DEN2, or DEN3 is designated by superscript. Lackof conservation is designated by no superscript. ^(b)Amino acid positionin DEN4 polyprotein beginning with the methionine residue of the Cprotein (nt 102-104) as residue #1. ^(c)not applicable

TABLE 33 Sequence analysis of rDEN2/4Δ30 clone 27(p4)-2-2A2. MutationNucleotide Gene Nucleotide Amino acid 743 M anchor G > A Gly > Glu 1493 E C > U Ser > Phe 7544* NS4B C > U Ala > Val *Same as DEN4 nucleotideposition 7546

TABLE 34 Sequence analysis of rDEN2/4Δ30 clone 27(p3)-2-1A1. MutationNucleotide Gene Nucleotide Amino acid 1345 E U > C Tyr > His  4885* NS3G > A Glu > Lys 8297 NS5 G > A Arg > Lys *Codon adjacent to 5-FUmutation 4891

TABLE 35 Recombinant virus rDEN2/4Δ30 bearing Vero adaptation mutationscan be recovery and titered on Vero cells. Virus titer in indicated cellline¹ Virus titer following (log₁₀PFU/ml) recovery in Vero Virus C6/36Vero cells (log₁₀PFU/ml) rDEN2/4Δ30 wt 5.2 1.7 <0.7 rDEN2/4Δ30-7153 5.45.2 <0.7 rDEN2/4Δ30-7162 5.4 5.3  nd² rDEN2/4Δ30-7182 4.7 4.9 2.3rDEN2/4Δ30-7630 5.3 4.8 1.3 rDEN2/4Δ30-7153-7163 5.1 4.7 ndrDEN2/4Δ30-7153-7182 4.1 3.2 nd rDEN2/4Δ30-7546-7630 5.2 5.2 nd ¹Virusrecovered following transfection of C6/36 mosquito cells was terminallydiluted once in C6/36 cells and titered simultaneously in C6/36 cellsand Vero cells. ²not determined

TABLE 36 Putative Vero cell adaptation mutations of dengue type 4 virusand the corresponding wildtype amino acid residue in other dengueviruses. Amino acid Mutant Amino acid in indicated wt dengue virus^(b)Mutation position^(a) residue DEN4 DEN1 DEN2 DEN3 1455 452 F V I A A2280 727 L F ^(c) F F F 4891 1597 T I V I I 4995 1632 P S S S N 71292343 L P P P P 7153 2351 A V F F L 7162 2354 S L V V V 7163 2354 F L V VV 7182 2361 S G G G G 7546 2482 V A L T V 7630 2510 R K S S K ^(a)Aminoacid position is given for the polyprotein of DEN4 ^(b)DEN4 = rDEN4(GenBank AF326825); DEN1 = Western pacific (GenBank DVU88535); DEN2 =New Guinea C (GenBank AF038403); DEN3 = H87 (GenBank M93130)^(c)Underlined nucleotides are shared between DEN4 and one or moreadditional DEN types.

TABLE 37 Mutations known to attenuate dengue type 4 virus and thecorresponding wildtype amino acid residue in other dengue virus. Aminoacid Mutant Amino acid in indicated wt dengue virus^(b) Mutationposition^(a) residue DEN4 DEN1 DEN2 DEN3 5-FU mutations 2650 850 S N^(d) N N N 3442 1114 G E E E E 3540 1147 K E E E E 3575 1158 I M L A M3771 1224 G R R K R 4062 1321 A T L A T 4306 1402 S N E D D 4891 1597 TI V I I 4896 1599 S A A A A 4907 1602 F L L L L 4995 1632 P S S S N 50971666 N D D D D 5695 1865 G D D D D 6259 2053 A V V V V  7129^(c)  2343 LP P P P 7849 2583 I N K N K 8092 2664 G E Q Q Q 10186  3362 T I I I I10634  3′ UTR — — — — — Charge-cluster- 22, 23 2509, 2510 AA RK KS KS RKto-alanine 23, 24 2510, 2511 AA KE SE SE KE mutations 157, 158 2644,2645 AA EE EE EA EE 200, 201 2687, 2688 AA KH KH KY KH 356, 357 2843,2844 AA KE KE KE KE 387, 388 2874, 2875 AA KK RN KK RN 436, 437 2923,2924 AA DK HR DK DK 524, 525 3011, 3012 AA KK KI KK KI 525, 526 3012,3013 AA KD IP KE IP 642, 643 3129, 3130 AA ER ER IA KK 654, 655 3141,3142 AA DR ER ER ER 808, 809 3295, 3296 AA ED ED ED ED 827, 828 3314,3315 AA DK DK DK DK 877, 878 3364, 3365 AA KE NE NE NE 878, 879 3365,3366 AA EE EN EE EE ^(a)Amino acid position is given for the polyproteinof DEN4 ^(b)DEN4 = rDEN4 (GenBank AF326825); DEN1 = Western pacific(GenBank U88535); DEN2 = New Guinea C (GenBank AF038403); DEN3 = H87(GenBank M93130) ^(c)This mutation results in decreased replication ofDEN4 in mosquitoes. ^(d)Underlined nucleotides are shared between DEN4and one or more additional DEN types.

APPENDIX 1 Sequence of recombinant dengue type 4 virus strain 2ALOCUS       AF375882     10649 bp ss-RNA    linear     VRL-19-SEP-2001DEFINITION  Dengue virus type 4 recombinant clone 2A, complete genome.ACCESSION   AF375822 VERSION     AF375882.1   GI: 14269097 KEYWORDS    .SOURCE      Dengue virus type 4.   ORGANISM  Dengue virys type 4            Viruses; ssRNA positive-strand viruses, no DNA stage;Flaviviridae;               Flavivirus; Dengue virus group.REFERENCE   1 (bases 1 to 10649)  AUTHORS   Blaney, J. E. Jr., Johnson, D. H., Firestone, C. Y. Hanson, C. T.,            Murphy, B. R. and Whitehead, S. S.  TITLE     Chemical Mutagenesis of Dengue Virus Type 4 Yields Mutant Viruses            Which Are Temperature Sensitive in Vero Cells or Human Liver Cells            and Attenuated in Mice  JOURNAL   J. Virol. 75 (20), 9731-9740 (2001)   MEDLINE   21443968   PUBMED   11559806 REFERENCE   2 (bases 1 to 10649)  AUTHORS   Blaney, J. E. Jr., Johnson, D. H., Firestone, C. Y. Hanson, C. T.,            Murphy, B. R. and Whitehead, S. S.  TITLE     Direct Submission  JOURNAL   Submitted (02-MAY-2001) LID, NIAID, 7 Center Drive, Bethesda, MD            20892, USA FEATURES             Location/Qualifiers     source          1 . . . 10649                                 /organism = “Dengue virus type 4”                     /virion                      /db_xref =“taxon: 11070”      mat_peptide     102 . . . 440                     /note = “anchC”                      /product =“anchored capsid protein”      mat_peptide     102 . . . 398                     /note = “virC”                      /product =“virion capsid protein”      CDS             102 . . . 10265                     /codon_start = 1                      /product =“polyprotein precursor”                      /protein_id = “AAK58017.1”                     /db_xref = “GI: 14269098” /translation =“MNQRKKVVRPPFNMLKRERNRVSTPQGLVKRFSTGLFSGKGPLRMVLAFITFLRVLSIPPTAGILKRWGQLKKNKAIKILIGFRKEIGRMLNILNGRKRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILRNPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSCSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTAMITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGNSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLVLWIGTNSRNTSMAMTCIAVGGITLFLGFTVQADMGCVVSWSGKELKCGSGIFVVDNVHTWTEQYKFQPESPARLASAILNAHKDGVCGIRSTTRLENVMWKQITNELNYVLWEGGHDLTVVAGDVKGVLTKGKRALTPPVSDLKYSWKTWGKAKIFTPEARNSTFLIDGPDTSECPNERRAWNSLEVEDYGFGMFTTNIWMKFREGSSEVCDHRLMSAAIKDQKAVHADMGYWIESSKNQTWQIEKASLIEVKTCLWPKTHTLWSNGVLESQMLIPKSYAGPFSQHNYRQGYATQTVGPWHLGKLEIDFGECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCRSCTMPPLRFLGEDGCWYGMEIRPLSEKEENMVKSQVTAGQGTSETFSMGLLCLTLFVEECLRRRVTRKHMILVVVITLCAIILGGLTWMDLLRALIMLGDTMSGRIGGQIHLAIMAVFKMSPGYVLGVFLRKLTSRETALMVIGMAMTTVLSIPHDLMELIDGISLGLILLKIVTQFDNTQVGTLALSLTFIRSTMPLVMAWRTIMAVLFVVTLIPLCRTSCLQKQSHWVEITALILGAQALPVYLMTLMKGASRRSWPLNEGIMAVGLVSLLGSALLKNDVPLAGPMVAGGLLLAAYVMSGSSADLSLEKAANVQWDEMADITGSSPIIEVKQDEDGSFSIRDVEETNMITLLVKLALITVSGLYPLAIPVTMTLWYMWQVKTQRSGALWDVPSPAATKKAALSEGVYRIMQRGLFGKTQVGVGIHMEGVFHTMWHVTRGSVICHETGRLEPSWADVRNDMISYGGGWRLGDKWDKEEDVQVLAIEPGKNPKHVQTKPGLFKTLTGEIGAVTLDFKPGTSGSPIINRKGKVIGLYGNGVVTKSGDYVSAITQAERIGEPDYEVDEDIFRKKRLTIMDLHPGAGKTKRILPSIVREALKRRLRTLILAPTRVVAAEMEEALRGLPIRYQTPAVKSEHTGREIVDLMCHATFTTRLLSSTRVPNYNLIVMDEAHFTDPSSVAARGYISTRVEMGEAAAIFMTATPPGATDPFPQSNSPIEDIEREIPERSWNTGFDWITDYQGKTVWFVPSIKAGNDIANCLRKSGKKVIQLSRKTFDTEYPKTKLTDWDFVVTTDISEMGANFRAGRVIDPRRCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYVFSGDPLKNDEDHAHWTEAKMLLDNIYTPEGIIPTLFGPEREKTQAIDGEFRLRGEQRKTFVELMRRGDLPVWLSYKVASAGISYKDREWCFTGERNNQILEENMEVEIWTREGEKKKLRPRWLDARVYADPMALKDFKEFASGRKSITLDILTEIASLPTYLSSRAKLALDNIVMLHTTERGGRAYQHALNELPESLETLMLVALLGAMTAGIFLFFMQGKGIGKLSMGLITIAVASGLLWVAEIQPQWIAASIILEFFLMVLLIPEPEKQRTPQDNQLIYVILTILTIIGLIAANEMGLIEKTKTDFGFYQVKTETTILDVDLRPASAWTLYAVATTILTPMLRHTIENTSANLSLAAIANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVNPTTLTASLVMLLVHYAIIGPGLQAKATREAQKRTAAGIMKHPTVDGITVIDLEPISYDPKFEKQLGQVMLLVLCAGQLLLMRTTWAFCEVLTLATGPILTLWEGNPGRFWNTTIAVSTANIFRGSYLAGAGLAFSLIKNAQTPRRGTGTTGETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGSKIKHAVSRGSSKIRWIVERGMVKPKGKVVDLGCGRGGWSYYMATLKNVTEVKGYTKGGPGHEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTIEEGRTLRVLKMVEPWLSSKPEFCIKVLNPYMPTVIEELEKLQRKHGGNLVRCPLSRNSTHEMYWVSGASGNIVSSVNTTSKMLLNRFTTRHRKPTYEKDVDLGAGTRSVSTETEKPDMTIIGRRLQRLQEEHKETWHYDQENPYRTWAYHGSYEAPSTGSASSMVNGVVKLLTKPWDVIPMVTQLAMTDTTPFGQQRVFKEKVDTRTPQPKPGTRMVMTTTANWLWALLGKKKNPRLCTREEFISKVRSNAAIGAVFQEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMGKREKKLGEFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSWSGVEGEGLHRLGYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQMAPHHKILAKAIFKLTYQNKVVKVLRPTPRGAVMDIISRKDQRGSGQVGTYGLNTFTNMEVQLIRQMEAEGVITQDDMQNPKGLKERVEKWLKECGVDRLKRMAISGDDCVVKPLDERFGTSLLFLNDMGKVRKDIPQWEPSKGWKNWQEVPFCSHHFHKIFMKDGRSLVVPCRNQDELIGRARISQGAGWSLRETACLGKAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWSIHAHHQWMTTEDMLKVWNRVWIEDNPNMTDKTPVHSWEDIPYLGKREDLWCGSLIGLSSRATWAXNIHTAITQVRNLIGKEEYVDYMPVMKRYSAPSESEGVL”     mat_peptide     441 . . . 938                      /note = “prM”                     /product = “membrane precursor protein”     mat_peptide     714 . . . 938                      /note = “M”                     /product = “membrane protein”     mat_peptide     939 . . . 2423                      /note = “E”                     /product = “envelope protein”     mat_peptide     2424 . . . 3479                      /product =“NS1 protein”      mat_peptide     3480 . . . 4133                     /product = “NS2A protein”     mat_peptide     4134 . . . 4523                      /product =“NS2B protein”      mat_peptide     4524 . . . 6377                     /product = “NS3 protein”     mat_peptide     6378 . . . 6758                      /product =“NS4A protein”      mat_peptide     6759 . . . 6827                     /product = “2K protein”     mat_peptide     6828 . . .  7562                      /product =“NS4B protein”      mat_peptide     7563 . . . 10262                     /product = “NS5 protein”BASE COUNT    3302 a     2212 c    2800 g   2335 t ORIGIN    1 agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg cttaacacag   61 ttctaacagt ttgtttgaat agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg  121 tggttagacc acctttcaat atgctgaaac gcgagagaaa ccgcgtatc aacccctcaag  181 ggttggtgaa gagattctca accggacttt tttctgggaa aggaccctt acggatggtgc  241 tagcattcat cacgtttttg cgagtccttt ccatcccacc aacagcagg gattctgaaga  301 gatggggaca gttgaagaaa aataaggcca tcaagatact gattggatt caggaaggaga  361 taggccgcat gctgaacatc ttgaacggga gaaaaaggtc aacgataac attgctgtgct  421 tgattcccac cgtaatggcg ttttccttgt caacaagaga tggcgaacc cctcatgatag  481 tggcaaaaca tgaaaggggg agacctctct tgtttaagac aacagaggg gatcaacaaat  541 gcactctcat tgccatggac ttgggtgaaa tgtgtgagga cactgtcac gtataaatgcc  601 ccctactggt caataccgaa cctgaagaca ttgattgctg gtgcaacct cacgtctacct  661 gggtcatgta tgggacatgc acccagagcg gagaacggag acgagagaa gcgctcagtag  721 ctttaacacc acattcagga atgggattgg aaacaagagc tgagacatg gatgtcatcgg  781 aaggggcttg gaagcatgct cagagagtag agagctggat actcagaaa cccaggattcg  841 cgctcttggc aggatttatg gcttatatga ttgggcaaac aggaatcca gcgaactgtct  901 tctttgtcct aatgatgctg gtcgccccat cctacggaat gcgatgcgt aggagtaggaa  961 acagagactt tgtggaagga gtctcaggtg gagcatgggt cgacctggt gctagaacatg 1021 gaggatgcgt cacaaccatg gcccagggaa aaccaacctt ggattttgaa ctgactaaga 1081 caacagccaa ggaagtggct ctgttaagaa cctattgcat tgaagcctca atatcaaaca 1141 taactacggc aacaagatgt ccaacgcaag gagagcctta tctgaaagag gaacaggacc 1201 aacagtacat ttgccggaga gatgtggtag acagagggtg gggcaatggc tgtggcttgt 1261 ttggaaaagg aggagttgtg acatgtgcga agttttcatg ttcggggaag ataacaggca 1321 atttggtcca aattgagaac cttgaataca cagtggttgt aacagtccac aatggagaca 1381 cccatgcagt aggaaatgac acatccaatc atggagttac agccatgata actcccaggt 1441 caccatcggt ggaagtcaaa ttgccggact atggagaact aacactcgat tgtgaaccca 1501 ggtctggaat tgactttaat gagatgattc tgatgaaaat gaaaaagaaa acatggctcg 1561 tgcataagca atggtttttg gatctgcctc ttccatggac agcaggagca gacacatcag 1621 aggttcactg gaattacaaa gagagaatgg tgacatttaa ggttcctcat gccaagagac 1681 aggatgtgac agtgctggga tctcaggaag gagccatgca ttctgccctc gctggagcca 1741 cagaagtgga ctccggtgat ggaaatcaca tgtttgcagg acatcttaag tgcaaagtcc 1801 gtatggagaa attgagaatc aagggaatgt catacacgat gtgttcagga aagttttcaa 1861 ttgacaaaga gatggcagaa acacagcatg ggacaacagt ggtgaaagtc aagtatgaag 1921 gtgctggagc tccgtgtaaa gtccccatag agataagaga tgtaaacaag gaaaaagtgg 1981 ttgggcgtat catctcatcc acccctttgg ctgagaatac caacagtgta accaacatag 2041 aattagaacc cccctttggg gacagctaca tagtgatagg tgttggaaac agcgcattaa 2101 cactccattg gttcaggaaa gggagttcca ttggcaagat gtttgagtcc acatacagag 2161 gtgcaaaacg aatggccatt ctaggtgaaa cagcttggga ttttggttcc gttggtggac 2221 tgttcacatc attgggaaag gctgtgcacc aggtttttgg aagtgtgtat acaaccatgt 2281 ttggaggagt ctcatggatg attagaatcc taattgggtt cttagtgttg tggattggca 2341 cgaactcaag gaacacttca atggctatga cgtgcatagc tgttggagga atcactctgt 2401 ttctgggctt cacagttcaa gcagacatgg gttgtgtggt gtcatggagt gggaaagaat 2461 tgaagtgtgg aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca 2521 aatttcaacc agagtcccca gcgagactag cgtctgcaat attaaatgcc cacaaagatg 2581 gggtctgtgg aattagatca accacgaggc tggaaaatgt catgtggaag caaataacca 2641 acgagctaaa ctatgttctc tgggaaggag gacatgacct cactgtagtg gctggggatg 2701 tgaagggggt gttgaccaaa ggcaagagag cactcacacc cccagtgagt gatctgaaat 2761 attcatggaa gacatgggga aaagcaaaaa tcttcacccc agaagcaaga aatagcacat 2821 ttttaataga cggaccagac acctctgaat gccccaatga acgaagagca tggaactctc 2881 ttgaggtgga agactatgga tttggcatgt tcacgaccaa catatggatg aaattccgag 2941 aaggaagttc agaagtgtgt gaccacaggt taatgtcagc tgcaattaaa gatcagaaag 3001 ctgtgcatgc tgacatgggt tattggatag agagctcaaa aaaccagacc tggcagatag 3061 agaaagcatc tcttattgaa gtgaaaacat gtctgtggcc caagacccac acactgtgga 3121 gcaatggagt gctggaaagc cagatgctca ttccaaaatc atatgcgggc cctttttcac 3181 agcacaatta ccgccagggc tatgccacgc aaaccgtggg cccatggcac ttaggcaaat 3241 tagagataga ctttggagaa tgccccggaa caacagtcac aattcaggag gattgtgacc 3301 atagaggccc atctttgagg accaccactg catctggaaa actagtcacg caatggtgct 3361 gccgctcctg cacgatgcct cccttaaggt tcttgggaga agatgggtgc tggtatggga 3421 tggagattag gcccttgagt gaaaaagaag agaacatggt caaatcacag gtgacggccg 3481 gacagggcac atcagaaact ttttctatgg gtctgttgtg cctgaccttg tttgtggaag 3541 aatgcttgag gagaagagtc actaggaaac acatgatatt agttgtggtg atcactcttt 3601 gtgctatcat cctgggaggc ctcacatgga tggacttact acgagccctc atcatgttgg 3661 gggacactat gtctggtaga ataggaggac agatccacct agccatcatg gcagtgttca 3721 agatgtcacc aggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag 3781 cactaatggt aataggaatg gccatgacaa cggtgctttc aattccacat gaccttatgg 3841 aactcattga tggaatatca ctgggactaa ttttgctaaa aatagtaaca cagtttgaca 3901 acacccaagt gggaacctta gctctttcct tgactttcat aagatcaaca atgccattgg 3961 tcatggcttg gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4021 ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag 4081 cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga agatcttggc 4141 ctcttaacga gggcataatg gctgtgggtt tggttagtct cttaggaagc gctcttttaa 4201 agaatgatgt ccctttagct ggcccaatgg tggcaggagg cttacttctg gcggcttacg 4261 tgatgagtgg tagctcagca gatctgtcac tagagaaggc cgccaacgtg cagtgggatg 4321 aaatggcaga cataacaggc tcaagcccaa tcatagaagt gaagcaggat gaagatggct 4381 ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg aaactggcac 4441 tgataacagt gtcaggtctc taccccttgg caattccagt cacaatgacc ttatggtaca 4501 tgtggcaagt gaaaacacaa agatcaggag ccctgtggga cgtcccctca cccgctgcca 4561 ctaaaaaagc cgcactgtct gaaggagtgt acaggatcat gcaaagaggg ttattcggga 4621 aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtaa 4681 caagaggatc agtgatctgc cacgagactg ggagattgga gccatcttgg gctgacgtca 4741 ggaatgacat gatatcatac ggtgggggat ggaggcttgg agacaaatgg gacaaagaag 4801 aagacgttca ggtcctcgcc atagaaccag gaaaaaatcc taaacatgtc caaacgaaac 4861 ctggcctttt caagacccta actggagaaa ttggagcagt aacattagat ttcaaacccg 4921 gaacgtctgg ttctcccatc atcaacagga aaggaaaagt catcggactc tatggaaatg 4981 gagtagttac caaatcaggt gattacgtca gtgccataac gcaagccgaa agaattggag 5041 agccagatta tgaagtggat gaggacattt ttcgaaagaa aagattaact ataatqgact 5101 tacaccccgg agctggaaag acaaaaagaa ttcttccatc aatagtgaga gaagccttaa 5161 aaaggaggct acgaactttg attttagctc ccacgagagt ggtggcggcc gagatggaag 5221 aggccctacg tggactgcca atccgttatc agaccccagc tgtgaaatca gaacacacag 5281 gaagagagat tgtagacctc atgtgtcatg caaccttcac aacaagactt ttgtcatcaa . 5341 ccagggttcc aaattacaac cttatagtga tggatgaagc acatttcacc gatccttcta 5401 gtgtcgcggc tagaggatac atctcgacca gggtggaaat gggagaggca gcagccatct 5461 tcatgaccgc aacccctccc ggagcgacag atccctttcc ccagagcaac agcccaatag 5521 aagacatcga gagggaaatt ccggaaaggt catggaacac agggttcgac tggataacag 5581 actaccaagg gaaaactgtg tggtttgttc ccagcataaa agctggaaat gacattgcaa 5641 attgtttgag aaagtcggga aagaaagtta tccagttgag taggaaaacc tttgatacag 5701 agtatccaaa aacgaaactc acggactggg actttgtggt cactacagac atatctgaaa 5761 tgggggccaa ttttagagcc gggagagtga tagaccctag aagatgcctc aagccagtta 5821 tcctaccaga tgggccagag agagtcattt tagcaggtcc tattccagtg actccagcaa 5881 gcgctgctca gagaagaggg cgaataggaa ggaacccagc acaagaagac gaccaatacg 5941 ttttctccgg agacccacta aaaaatgatg aagatcatgc ccactggaca gaagcaaaga 6001 tgctgcttga caatatctac accccagaag ggatcattcc aacattgttt ggtccggaaa 6061 gggaaaaaac ccaagccatt gatggagagt ttcgcctcag aggggaacaa aggaagactt 6121 ttgtggaatt aatgaggaga ggagaccttc cggtgtggct gagctataag gtagcttctg 6181 ctggcatttc ttacaaagat cgggaatggt gcttcacagg ggaaagaaat aaccaaattt 6241 tagaagaaaa catggaggtt gaaatttgga ctagagaggg agaaaagaaa aagctaaggc 6301 caagatggtt agatgcacgt gtatacgctg accccatggc tttgaaggat ttcaaggagt 6361 ttgccagtgg aaggaagagt ataactctcg acatcctaac agagattgcc agtttgccaa 6421 cttacctttc ctctagggcc aagctcgccc ttgataacat agtcatgctc cacacaacag 6481 aaagaggagg gagggcctat caacacgccc tgaacgaact tccggagtca ctggaaacac 6541 tcatgcttgt agctttacta ggtgctatga cagcaggcat cttcctgttt ttcatgcaag 6601 ggaaaggaat agggaaattg tcaatgggtt tgataaccat tgcggtggct agtggcttgc 6661 tctgggtagc agaaattcaa ccccagtgga tagcggcctc aatcatacta gagttttttc 6721 tcatggtact gttgataccg gaaccagaaa aacaaaggac cccacaagac aatcaattga 6781 tctacgtcat attgaccatt ctcaccatca ttggtctaat agcagccaac gagatggggc 6841 tgattgaaaa aacaaaaacg gattttgggt tttaccaggt aaaaacagaa accaccatcc 6901 tcgatgtgga cttgagacca gcttcagcat ggacgctcta tgcagtagcc accacaattc 6961 tgactcccat gctgagacac accatagaaa acacgtcggc caacctatct ctagcagcca 7021 ttgccaacca ggcagccgtc ctaatggggc ttggaaaagg atggccgctc cacagaatgg 7081 acctcggtgt gccgctgtta gcaatgggat gctattctca agtgaaccca acaaccttga 7141 cagcatcctt agtcatgctt ttagtccatt atgcaataat aggcccagga ttgcaggcaa 7201 aagccacaag agaggcccag aaaaggacag ctgctgggat catgaaaaat cccacagtgg 7261 acgggataac agtaatagat ctagaaccaa tatcctatga cccaaaattt gaaaagcaat 7321 tagggcaggt catgctacta gtcttgtgtg ctggacaact actcttgatg agaacaacat 7381 gggctttctg tgaagtcttg actttggcca caggaccaat cttgaccttg tgggagggca 7441 acccgggaag gttttggaac acgaccatag ccgtatccac cgccaacatt ttcaggggaa 7501 gttacttggc gggagctgga ctggcttttt cactcataaa gaatgcacaa acccctagga 7561 ggggaactgg gaccacagga gagacactgg gagagaagtg gaagagacag ctaaactcat 7621 tagacagaaa agagtttgaa gagtataaaa gaagtggaat actagaagtg gacaggactg 7681 aagccaagtc tgccctgaaa gatgggtcta aaatcaagca tgcagtatct agagggtcca 7741 gtaagatcag atggattgtt gagagaggga tggtaaagcc aaaagggaaa gttgtagatc 7801 ttggctgtgg gagaggagga tggtcttatt acatggcgac actcaagaac gtgactgaag 7861 tgaaagggta tacaaaagga ggtccaggac atgaagaacc gattcccatg gctacttatg 7921 gttggaattt ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc acagagcaag 7981 tggacaccct gctctgtgat attggggagt catcttctaa tccaacaata gaggaaggaa 8041 gaacattaag agttttgaag atggtggagc catggctctc ttcaaaacct gaattctgca 8101 tcaaagtcct taacccctac atgccaacag tcatagaaga gctggagaaa ctgcagagaa 8161 aacatggtgg gaaccttgtc agatgcccgc tgtccaggaa ctccacccat gagatgtatt 8221 gggtgtcagg agcgtcggga aacattgtga gctctgtgaa cacaacatca aagatgttgt 8281 tgaacaggtt cacaacaagg cataggaaac ccacttatga gaaggacgta gatcttgggg 8341 caggaacgag aagtgtctcc actgaaacag aaaaaccaga catgacaatc attgggagaa 8401 ggcttcagcg attgcaagaa gagcacaaag aaacctggca ttatgatcag gaaaacccat 8461 acagaacctg ggcgtatcat ggaagctatg aagctccttc gacaggctct gcatcctcca 8521 tggtgaacgg ggtggtaaaa ctgctaacaa aaccctggga tgtgattcca atggtgactc 8581 agttagccat gacagataca accccttttg ggcaacaaag agtgttcaaa gagaaggtgg 8641 ataccagaac accacaacca aaacccggta cacgaatggt tatgaccacg acagccaatt 8701 ggctgtgggc cctccttgga aagaagaaaa atcccagact gtgcacaagg gaagagttca 8761 tctcaaaagt tagatcaaac gcagccatag gcgcagtctt tcaggaagaa cagggatgga 8821 catcagccag tgaagctgtg aatgacagcc ggttttggga actggttgac aaagaaaggg 8881 ccctacacca ggaagggaaa tgtgaatcgt gtgtctataa catgatggga aaacgtgaga 8941 aaaagttagg agagtttggc agagccaagg gaagccgagc aatctggtac atgtggctgg 9001 gagcgcggtt tctggaattt gaagccctgg gttttttgaa tgaagatcac tggtttggca 9061 gagaaaattc atggagtgga gtggaagggg aaggtctgca cagattggga tatatcctgg 9121 aggagataga caagaaggat ggagacctaa tgtatgctga tgacacagca ggctgggaca 9181 caagaatcac tgaggatgac cttcaaaatg aggaactgat cacggaacag atggctcccc 9241 accacaagat cctagccaaa gccattttca aactaaccta tcaaaacaaa gtggtgaaag 9301 tcctcagacc cacaccgaga ggagcggtga tggatatcat atccaggaaa gaccaaagag 9361 gtagtggaca agttggaaca tatggtttga acacattcac caacatggaa gttcaactca 9421 tccgccaaat ggaagctgaa ggagtcatca cacaagatga catgcagaac ccaaaagggt 9481 tgaaagaaag agttgagaaa tggctgaaag agtgtggtgt cgacaggtta aagaggatgg 9541 caatcagtgg agacgattgc gtggtgaagc ccctagatga gaggtttggc acttccctcc 9601 tcttcttgaa cgacatggga aaggtgagga aagacattcc gcagtgggaa ccatctaagg 9661 gatggaaaaa ctggcaagag gttccttttt gctcccacca ctttcacaag atctttatga 9721 aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca 9781 gaatctcgca gggagctgga tggagcttaa gagaaacagc ctgcctgggc aaagcttacg 9841 cccagatgtg gtcgcttatg tacttccaca gaagggatct gcgtttagcc tccatggcca 9901 tatgctcagc agttccaacg gaatggtttc caacaagcag aacaacatgg tcaatccacg 9961 ctcatcacca gtggatgacc actgaagata tgctcaaagt gtggaacaga gtgtggatag10021 aagacaaccc taatatgact gacaagactc cagtccattc gtgggaagat ataccttacc10081 tagggaaaag agaggatttg tggtgtggat ccctgattgg actttcttcc agagccacct10141 gggcgaagaa cattcacacg gccataaccc aggtcaggaa cctgatcgga aaagaggaat10201 acgtggatta catgccagta atgaaaagat acagtgctcc ttcagagagt gaaggagttc10261 tgtaattacc aacaacaaac accaaaggct attgaagtca ggccacttgt gccacggttt10321 gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtaataat ccccagggag10381 gccatgcgcc acggaagctg tacgcgtggc atattggact agcggttaga ggagacccct10441 cccatcactg acaaaacgca gcaaaagggg gcccgaagcc aggaggaagc tgtactcctg10501 gtggaaggac tagaggttag aggagacccc cccaacacaa aaacagcata ttgacgctgg10561 gaaagaccag agatcctgct gtctctgcaa catcaatcca ggcacagagc gccgcaagat10621 ggattggtgt tgttgatcca acaggttct

APPENDIX 2 Sequence of recombinant dengue typ 4 virus strain rDEN4LOCUS       AF326825    10649 bp    RNA             VRL       03-JAN-2001DEFINITION  Dengue virus type 4 recombinant clone rDEN4, complete sequence.ACCESSION   AF326825 VERSION     AF326825.1   GI: 12018169 KEYWORDS    .SOURCE      Dengue virus type 4.   ORGANISM  Dengue virus type 4            Viruses; ssRNA positive-strand viruses, no DNA stage;Flaviviridae;             Flavivirus; Dengue virus group.REFERENCE   1 (bases 1 to 10649)  AUTHORS   Durbin, A. P., Karron, R. A., Sun, W., Vaughn, D. W., Reynolds, M. J.,            Perreault, J. R., Men, R. H., Lai, C. J., Elkins, W. R., Chanock, R. M.,            Murphy, B. R. and Whitehead, S. S.  TITLE     A live attenuated dengue virus type 4 vaccine candidate with a 30            nucleotide deletion in the 3′ untranslated region is highly            attenuated and immunogenic in humans   JOURNAL   UnpublishedREFERENCE   2 (bases 1 to 10649)   AUTHORS   Whitehead, S. S.  TITLE     Direct Submission  JOURNAL   Submitted (08-DEC-2000) LID, NIAID, 7 Center Drive, Bethesda, MD            20892, USA FEATURES             Location/Qualifiers     source          1 . . . 10649                      /organism =“Dengue virus type 4”                      /db_xref = “taxon: 11070”                     /clone = “rDEN4”      mat_peptide     102 . . . 440                     /product = “anchored capsid (anchC) protein”     mat_peptide     102 . . . 398                      /product =“virion capsid (virC) protein”      CDS             102 . . . 10265                     /codon_start = 1                      /product =“polyprotein precursor”                      /protein_id = “AAG45435.1”                     /db_xref = “GI: 12018170” /translation =“MNQRKKVVRPPFNMLKRERNRVSTPQGLVKRFSTGLFSGKGPLRMVLAFITFLRVLSIPPTAGILKRWGQLKKNKAIKILIGFRKEIGRMLNILNGRKRSTITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILRNPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFSCSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTAMITPRSPSVEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEVHWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGMHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTVVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGNSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLVLWIGTNSRNTSMAMTCIAVGGITLFLGFTVQADMGCVASWSGKELKCGSGIFVVDNVHTWTEQYKFQPESPARLASAILNAHKDGVCGIRSTTRLENVMWKQITNELNYVLWEGGHDLTVVAGDVKGVLTKGKRALTPPVSDLKYSWKTWGKAKIFTPEARNSTFLIDGPDTSECPNERRAWNSLEVEDYGFGMFTTNIWMKFREGSSEVCDHRLMSAAIKDQKAVHADMGYWIESSKNQTWQIEKASLIEVKTCLWPKTHTLWSNGVLESQMLIPKSYAGPFSQHNYRQGYATQTVGPWHLGKLEIDFGECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCRSCTMPPLRFLGEDGCWYGMEIRPLSEKEENMVKSQVTAGQGTSETFSMGLLCLTLFVEECLRRRVTRKHMILVVVITLCAIILGGLTWMDLLRALIMLGDTMSGRIGGQIHLAIMAVFKMSPGYVLGVFLRKLTSRETALMVIGMAMTTVLSIPHDLMELIDGISLGLILLKIVTQFDNTQVGTLALSLTFIRSTMPLVMAWRTIMAVLFVVTLIPLCRTSCLQKQSHWVEITALILGAQALPVYLMTLMKGASRRSWPLNEGIMAVGLVSLLGSALLKNDVPLAGPMVAGGLLLAAYVMSGSSADLSLEKAANVQWDEMADITGSSPIVEVKQDEDGSFSIRDVEETNMITLLVKLALITVSGLYPLAIPVTMTLWYMWQVKTQRSGALWDVPSPAATKKAALSEGVYRIMQRGLFGKTQVGVGIHMEGVFHTMWHVTRGSVICHETGRLEPSWADVRNDMISYGGGWRLGDKWDKEEDVQVLAIEPGKNPKHVQTKPGLFKTLTGEIGAVTLDFKPGTSGSPIINRKGKVIGLYGNGVVTKSGDYVSAITQAERIGEPDYEVDEDIFRKKRLTIMDLHPGAGKTKRILPSIVREALKRRLRTLILAPTRVVAAEMEEALRGLPIRYQTPAVKSEHTGREIVDLMCHATFTTRLLSSTRVPNYNLIVMDEAHFTDPSSVAARGYISTRVEMGEAAAIFMTATPPGATDPFPQSNSPIEDIEREIPERSWNTGFDWITDYQGKTVWFVPSIKAGNDIANCLRKSGKKVIQLSRKTFDTEYPKTKLTDWDFVVTTDISEMGANFRAGRVIDPRRCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYVFSGDPLKNDEDHAHWTEAKMLLDNIYTPEGIIPTLFGPEREKTQAIDGEFRLRGEQRKTFVELMRRGDLPVWLSYKVASAGISYEDREWCFTGERNNQILEENMEVEIWTREGEKKKLRPRWLDARVYADPMALKDFKEFASGRKSITLDILTEIASLPTYLSSRAKLALDNIVMLHTTERGGRAYQHALNELPESLETLMLVALLGAMTAGIFLFFMQGKGIGKLSMGLITIAVASGLLWVAEIQPQWIAASIILEFFLMVLLIPEPEKQRTPQDNQLIYVILTILTIIGLIAANEMGLIEKTKTDFGFYQVKTETTILDVDLRPASAWTLYAVATTILTPMLRHTIENTSANLSLAAIANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVNPTTLTASLVMLLVHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGITVIDLEPISYDPKFEKQLGQVMLLVLCAGQLLLMRTTWAFCEVLTLATGPILTLWEGNPGRFWNTTIAVSTANIFRGSYLAGAGLAFSLIKNAQTPRRGTGTTGETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGSKIKHAVSRGSSKIRWIVERGMVKPKGKVVDLGCGRGGWSYYMATLKNVTEVKGYTKGGPGHEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTIEEGRTLRVLKMVEPWLSSKPEFCIKVLNPYMPTVIEELEKLQRKHGGNLVRCPLSRNSTHEMYWVSGASGNIVSSVNTTSKMLLNRFTTRHRKPTYEKDVDLGAGTRSVSTETEKPDMTIIGRRLQRLQEEHKETWHYDQENPYRTWAYHGSYEAPSTGSASSMVNGVVKLLTKPWDVIPMVTQLAMTDTTPFGQQRVFKEKVDTRTPQPKPGTRMVMTTTANWLWALLGKKKNPRLCTREEFISKVRSNAAIGAVFQEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMGKREKKLGEFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSWSGVEGEGLHRLGYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQMAPHHKILAKAIFKLTYQNKVVKVLRPTPRGAVMDIISRKDQRGSGQVGTYGLNTFTNMEVQLIRQMEAEGVITQDDMQNPKGLKERVEKWLKECGVDRLKRMAISGDDCVVKPLDERFGTSLLFLNDMGKVRKDIPQWEPSKGWKNWQEVPFCSHHFHKIFMKDGRSLVVPCRNQDELIGRARISQGAGWSLRETACLGKAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWSIHAHHQWMTTEDMLKVWNRVWIEDNPNMTDKTPVHSWEDIPYLGKREDLWCGSLIGLSSRATWAKNIHTAITQVRNLIGKEEYVDYMPVMKRYSAPSESEGVL”     mat_peptide     441 . . . 938                      /product =“membrane precursor (prM) protein”      mat_peptide     714 . . . 938                     /product = “membrane (M) protein”     mat_peptide     939 . . . 2423                      /product =“envelope (E) protein”      mat_peptide     2424 . . . 3479                     /product = “NS1 protein”     mat_peptide     3480 . . . 4133                      /product =“NS2A protein”      mat_peptide     4134 . . . 4523                     /product = “NS2B protein”     mat_peptide     4524 . . . 6377                      /product =“NS3 protein”      mat_peptide     6378 . . . 6758                     /product = “NS4A protein”     mat_peptide     6759 . . . 6827                      /product =“2K protein”      mat_peptide     6828 . . . 7562                     /product = “NS4B protein”     mat_peptide     7563 . . . 10262                      /product =“NS5 protein” rDEN4 sequence    1 agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg cttaacacag   61 ttctaacagt ttgtttgaat agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg  121 tggttagacc acctttcaat atgctgaaac gcgagagaaa ccgcgtatca acccctcaag  181 ggttggtgaa gagattctca accggacttt tttctgggaa aggaccctta cggatggtgc  241 tagcattcat cacgtttttg cgagtccttt ccatcccacc aacagcaggg attctgaaga  301 gatggggaca gttgaagaaa aataaggcca tcaagatact gattggattc aggaaggaga  361 taggccgcat gctgaacatc ttgaacggga gaaaaaggtc aacgataaca ttgctgtgct  421 tgattcccac cgtaatggcg ttttccctca gcacaagaga tggcgaaccc ctcatgatag  481 tggcaaaaca tgaaaggggg agacctctct tgtttaagac aacagagggg atcaacaaat  541 gcactctcat tgccatggac ttgggtgaaa tgtgtgagga cactgtcacg tataaatgcc  601 ccctactggt caataccgaa cctgaagaca ttgattgctg gtgcaacctc acgtctacct  661 gggtcatgta tgggacatgc acccagagcg gagaacggag acgagagaag cgctcagtag  721 ctttaacacc acattcagga atgggattgg aaacaagagc tgagacatgg atgtcatcgg  781 aaggggcttg gaagcatgct cagagagtag agagctggat actcagaaac ccaggattcg  841 cgctcttggc aggatttatg gcttatatga ttgggcaaac aggaatccag cgaactgtct  901 tctttgtcct aatgatgctg gtcgccccat cctacggaat gcgatgcgta ggagtaggaa  961 acagagactt tgtggaagga gtctcaggtg gagcatgggt cgacctggtg ctagaacatg 1021 gaggatgcgt cacaaccatg gcccagggaa aaccaacctt ggattttgaa ctgactaaga 1081 caacagccaa ggaagtggct ctgttaagaa cctattgcat tgaagcctca atatcaaaca 1141 taactacggc aacaagatgt ccaacgcaag gagagcctta tctgaaagag gaacaggacc 1201 aacagtacat ttgccggaga gatgtggtag acagagggtg gggcaatggc tgtggcttgt 1261 ttggaaaagg aggagttgtg acatgtgcga agttttcatg ttcggggaag ataacaggca 1321 atttggtcca aattgagaac cttgaataca cagtggttgt aacagtccac aatggagaca 1381 cccatgcagt aggaaatgac acatccaatc atggagttac agccatgata actcccaggt 1441 caccatcggt ggaagtcaaa ttgccggact atggagaact aacactcgat tgtgaaccca 1501 ggtctggaat tgactttaat gagatgattc tgatgaaaat gaaaaagaaa acatggctcg 1561 tgcataagca atggtttttg gatctgcctc ttccatggac agcaggagca gacacatcag 1621 aggttcactg gaattacaaa gagagaatgg tgacatttaa ggttcctcat gccaagagac 1681 aggatgtgac agtgctggga tctcaggaag gagccatgca ttctgccctc gctggagcca 1741 cagaagtgga ctccggtgat ggaaatcaca tgtttgcagg acatcttaag tgcaaagtcc 1801 gtatggagaa attgagaatc aagggaatgt catacacgat gtgttcagga aagttttcaa 1861 ttgacaaaga gatggcagaa acacagcatg ggacaacagt ggtgaaagtc aagtatgaag 1921 gtgctggagc tccgtgtaaa gtccccatag agataagaga tgtaaacaag gaaaaagtgg 1981 ttgggcgtat catctcatcc acccctttgg ctgagaatac caacagtgta accaacatag 2041 aattagaacc cccctttggg gacagctaca tagtgatagg tgttggaaac agcgcattaa 2101 cactccattg gttcaggaaa gggagttcca ttggcaagat gtttgagtcc acatacagag 2161 gtgcaaaacg aatggccatt ctaggtgaaa cagcttggga ttttggttcc gttggtggac 2221 tgttcacatc attgggaaag gctgtgcacc aggtttttgg aagtgtgtat acaaccatgt 2281 ttggaggagt ctcatggatg attagaatcc taattgggtt cttagtgttg tggattggca 2341 cgaactcgag gaacacttca atggctatga cgtgcatagc tgttggagga atcactctgt 2401 ttctgggctt cacagttcaa gcagacatgg gttgtgtggc gtcatggagt gggaaagaat 2461 tgaagtgtgg aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca 2521 aatttcaacc agagtcccca gcgagactag cgtctgcaat attaaatgcc cacaaagatg 2581 gggtctgtgg aattagatca accacgaggc tggaaaatgt catgtggaag caaataacca 2641 acgagctaaa ctatgttctc tgggaaggag gacatgacct cactgtagtg gctggggatg 2701 tgaagggggt gttgaccaaa ggcaagagag cactcacacc cccagtgagt gatctgaaat 2761 attcatggaa gacatgggga aaagcaaaaa tcttcacccc agaagcaaga aatagcacat 2821 ttttaataga cggaccagac acctctgaat gccccaatga acgaagagca tggaactctc 2881 ttgaggtgga agactatgga tttggcatgt tcacgaccaa catatggatg aaattccgag 2941 aaggaagttc agaagtgtgt gaccacaggt taatgtcagc tgcaattaaa gatcagaaag 3001 ctgtgcatgc tgacatgggt tattggatag agagctcaaa aaaccagacc tggcagatag 3061 agaaagcatc tcttattgaa gtgaaaacat gtctgtggcc caagacccac acactgtgga 3121 gcaatggagt gctggaaagc cagatgctca ttccaaaatc atatgcgggc cctttttcac 3181 agcacaatta ccgccagggc tatgccacgc aaaccgtggg cccatggcac ttaggcaaat 3241 tagagataga ctttggagaa tgccccggaa caacagtcac aattcaggag gattgtgacc 3301 atagaggccc atctttgagg accaccactg catctggaaa actagtcacg caatggtgct 3361 gccgctcctg cacgatgcct cccttaaggt tcttgggaga agatgggtgc tggtatggga 3421 tggagattag gcccttgagt gaaaaagaag agaacatggt caaatcacag gtgacggccg 3481 gacagggcac atcagaaact ttttctatgg gtctgttgtg cctgaccttg tttgtggaag 3541 aatgcttgag gagaagagtc actaggaaac acatgatatt agttgtggtg atcactcttt 3601 gtgctatcat cctgggaggc ctcacatgga tggacttact acgagccctc atcatgttgg 3661 gggacactat gtctggtaga ataggaggac agatccacct agccatcatg gcagtgttca 3721 agatgtcacc aggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag 3781 cactaatggt aataggaatg gccatgacaa cggtgctttc aattccacat gaccttatgg 3841 aactcattga tggaatatca ctgggactaa ttttgctaaa aatagtaaca cagtttgaca 3901 acacccaagt gggaacctta gctctttcct tgactttcat aagatcaaca atgccattgg 3961 tcatggcttg gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4021 ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag 4081 cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga agatcttggc 4141 ctcttaacga gggcataatg gctgtgggtt tggttagtct cttaggaagc gctcttttaa 4201 agaatgatgt ccctttagct ggcccaatgg tggcaggagg cttacttctg gcggcttacg 4261 tgatgagtgg tagctcagca gatctgtcac tagagaaggc cgccaacgtg cagtgggatg 4321 aaatggcaga cataacaggc tcaagcccaa tcgtagaagt gaagcaggat gaagatggct 4381 ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg aaactggcac 4441 tgataacagt gtcaggtctc taccccttgg caattccagt cacaatgacc ttatggtaca 4501 tgtggcaagt gaaaacacaa agatcaggag ccctgtggga cgtcccctca cccgctgcca 4561 ctaaaaaagc cgcactgtct gaaggagtgt acaggatcat gcaaagaggg ttattcggga 4621 aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtaa 4681 caagaggatc agtgatctgc cacgagactg ggagattgga gccatcttgg gctgacgtca 4741 ggaatgacat gatatcatac ggtgggggat ggaggcttgg agacaaatgg gacaaagaag 4801 aagacgttca ggtcctcgcc atagaaccag gaaaaaatcc taaacatgtc caaacgaaac 4861 ctggcctttt caagacccta actggagaaa ttggagcagt aacattagat ttcaaacccg 4921 gaacgtctgg ttctcccatc atcaacagga aaggaaaagt catcggactc tatggaaatg 4981 gagtagttac caaatcaggt gattacgtca gtgccataac gcaagccgaa agaattggag 5041 agccagatta tgaagtggat gaggacattt ttcgaaagaa aagattaact ataatggact 5101 tacaccccgg agctggaaag acaaaaagaa ttcttccatc aatagtgaga gaagccttaa 5161 aaaggaggct acgaactttg attttagctc ccacgagagt ggtggcggcc gagatggaag 5221 aggccctacg tggactgcca atccgttatc agaccccagc tgtgaaatca gaacacacag 5281 gaagagagat tgtagacctc atgtgtcatg caaccttcac aggaagactt ttgtcatcaa 5341 ccagggttcc aaattacaac cttatagtga tggatgaagc acatttcacc gatccttcta 5401 gtgtcgcggc tagaggatac atctcgacca gggtggaaat gggagaggca gcagccatct 5461 tcatgaccgc aacccctccc ggagcgacag atccctttcc ccagagcaac agcccaatag 5521 aagacatcga gagggaaatt ccggaaaggt catggaacac agggttcgac tggataacag 5581 actaccaagg gaaaactgtg tggtttgttc ccagcataaa agctggaaat gacattgcaa 5641 attgtttgag aaagtcggga aagaaagtta tccagttgag taggaaaacc tttgatacag 5701 agtatccaaa aacgaaactc acggactggg actttgtggt cactacagac atatctgaaa 5761 tgggggccaa ttttagagcc gggagagtga tagaccctag aagatgcctc aagccagtta 5821 tcctaccaga tgggccagag agagtcattt tagcaggtcc tattccagtg actccagcaa 5881 gcgctgctca gagaagaggg cgaataggaa ggaacccagc acaagaagac gaccaatacg 5941 ttttctccgg agacccacta aaaaatgatg aagatcatgc ccactggaca gaagcaaaga 6001 tgctgcttga caatatctac accccagaag ggatcattcc aacattgttt ggtccggaaa 6061 gggaaaaaac ccaagccatt gatggagagt ttcgcctcag aggggaacaa aggaagactt 6121 ttgtggaatt aatgaggaga ggagaccttc cggtgtggct gagctataag gtagcttctg 6181 ctggcatttc ttacgaagat cgggaatggt gcttcacagg ggaaagaaat aaccaaattt 6241 tagaagaaaa catggaggtt gaaatttgga ctagagaggg agaaaagaaa aagctaaggc 6301 caagatggtt agatgcacgt gtatacgctg accccatggc tttgaaggat ttcaaggagt 6361 ttgccagtgg aaggaagagt ataactctcg acatcctaac agagattgcc agtttgccaa 6421 cttacctttc ctctagggcc aagctcgccc ttgataacat agtcatgctc cacacaacag 6481 aaagaggagg gagggcctat caacacgccc tgaacgaact tccggagtca ctggaaacac 6541 tcatgcttgt agctttacta ggtgctatga cagcaggcat cttcctgttt ttcatgcaag 6601 ggaaaggaat agggaaattg tcaatgggtt tgataaccat tgcggtggct agtggcttgc 6661 tctgggtagc agaaattcaa ccccagtgga tagcggcctc aatcatacta gagttttttc 6721 tcatggtact gttgataccg gaaccagaaa aacaaaggac cccacaagac aatcaattga 6781 tctacgtcat attgaccatt ctcaccatca ttggtctaat agcagccaac gagatggggc 6841 tgattgaaaa aacaaaaacg gattttgggt tttaccaggt aaaaacagaa accaccatcc 6901 tcgatgtgga cttgagacca gcttcagcat ggacgctcta tgcagtagcc accacaattc 6961 tgactcccat gctgagacac accatagaaa acacgtcggc caacctatct ctagcagcca 7021 ttgccaacca ggcagccgtc ctaatggggc ttggaaaagg atggccgctc cacagaatgg 7081 acctcggtgt gccgctgtta gcaatgggat gctattctca agtgaaccca acaaccttga 7141 cagcatcctt agtcatgctt ttagtccatt atgcaataat aggcccagga ttgcaggcaa 7201 aagccacaag agaggcccag aaaaggacag ctgctgggat catgaaaaat cccacagtgg 7261 acgggataac agtaatagat ctagaaccaa tatcctatga cccaaaattt gaaaagcaat 7321 tagggcaggt catgctacta gtcttgtgtg ctggacaact actcttgatg agaacaacat 7381 gggctttctg tgaagtcttg actttggcca caggaccaat cttgaccttg tgggagggca 7441 acccgggaag gttttggaac acgaccatag ccgtatccac cgccaacatt ttcaggggaa 7501 gttacttggc gggagctgga ctggcttttt cactcataaa gaatgcacaa acccctagga 7561 ggggaactgg gaccacagga gagacactgg gagagaagtg gaagagacag ctaaactcat 7621 tagacagaaa agagtttgaa gagtataaaa gaagtggaat actagaagtg gacaggactg 7681 aagccaagtc tgccctgaaa gatgggtcta aaatcaagca tgcagtatca agagggtcca 7741 gtaagatcag atggattgtt gagagaggga tggtaaagcc aaaagggaaa gttgtagatc 7801 ttggctgtgg gagaggagga tggtcttatt acatggcgac actcaagaac gtgactgaag 7861 tgaaagggta tacaaaagga ggtccaggac atgaagaacc gattcccatg gctacttatg 7921 gttggaattt ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc acagagcaag 7981 tggacaccct gctctgtgat attggggagt catcttctaa tccaacaata gaggaaggaa 8041 gaacattaag agttttgaag atggtggagc catggctctc ttcaaaacct gaattctgca 8101 tcaaagtcct taacccctac atgccaacag tcatagaaga gctggagaaa ctgcagagaa 8161 aacatggtgg gaaccttgtc agatgcccgc tgtccaggaa ctccacccat gagatgtatt 8221 gggtgtcagg agcgtcggga aacattgtga gctctgtgaa cacaacatca aagatgttgt 8281 tgaacaggtt cacaacaagg cataggaaac ccacttatga gaaggacgta gatcttgggg 8341 caggaacgag aagtgtctcc actgaaacag aaaaaccaga catgacaatc attgggagaa 8401 ggcttcagcg attgcaagaa gagcacaaag aaacctggca ttatgatcag gaaaacccat 8461 acagaacctg ggcgtatcat ggaagctatg aagctccttc gacaggctct gcatcctcca 8521 tggtgaacgg ggtggtaaaa ctgctaacaa aaccctggga tgtgattcca atggtgactc 8581 agttagccat gacagataca accccttttg ggcaacaaag agtgttcaaa gagaaggtgg 8641 ataccagaac accacaacca aaacccggta cacgaatggt tatgaccacg acagccaatt 8701 ggctgtgggc cctccttgga aagaagaaaa atcccagact gtgcacaagg gaagagttca 8761 tctcaaaagt tagatcaaac gcagccatag gcgcagtctt tcaggaagaa cagggatgga 8821 catcagccag tgaagctgtg aatgacagcc ggttttggga actggttgac aaagaaaggg 8881 ccctacacca ggaagggaaa tgtgaatcgt gtgtctataa catgatggga aaacgtgaga 8941 aaaagttagg agagtttggc agagccaagg gaagccgagc aatctggtac atgtggctgg 9001 gagcgcggtt tctggaattt gaagccctgg gttttttgaa tgaagatcac tggtttggca 9061 gagaaaattc atggagtgga gtggaagggg aaggtctgca cagattggga tatatcctgg 9121 aggagataga caagaaggat ggagacctaa tgtatgctga tgacacagca ggctgggaca 9181 caagaatcac tgaggatgac cttcaaaatg aggaactgat cacggaacag atggctcccc 9241 accacaagat cctagccaaa gccattttca aactaaccta tcaaaacaaa gtggtgaaag 9301 tcctcagacc cacaccgcgg ggagcggtga tggatatcat atccaggaaa gaccaaagag 9361 gtagtggaca agttggaaca tatggtttga acacattcac caacatggaa gttcaactca 9421 tccgccaaat ggaagctgaa ggagtcatca cacaagatga catgcagaac ccaaaagggt 9481 tgaaagaaag agttgagaaa tggctgaaag agtgtggtgt cgacaggtta aagaggatgg 9541 caatcagtgg agacgattgc gtggtgaagc ccctagatga gaggtttggc acttccctcc 9601 tcttcttgaa cgacatggga aaggtgagga aagacattcc gcagtgggaa ccatctaagg 9661 gatggaaaaa ctggcaagag gttccttttt gctcccacca ctttcacaag atctttatga 9721 aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca 9781 gaatctcgca gggagctgga tggagcttaa gagaaacagc ctgcctgggc aaagcttacg 9841 cccagatgtg gtcgcttatg tacttccaca gaagggatct gcgtttagcc tccatggcca 9901 tatgctcagc agttccaacg gaatggtttc caacaagcag aacaacatgg tcaatccacg 9961 ctcatcacca gtggatgacc actgaagata tgctcaaagt gtggaacaga gtgtggatag10021 aagacaaccc taatatgact gacaagactc cagtccattc gtgggaagat ataccttacc10081 tagggaaaag agaggatttg tggtgtggat ccctgattgg actttcttcc agagccacct10141 gggcgaagaa cattcatacg gccataaccc aggtcaggaa cctgatcgga aaagaggaat10201 acgtggatta catgccagta atgaaaagat acagtgctcc ttcagagagt gaaggagttc10261 tgtaattacc aacaacaaac accaaaggct attgaagtca ggccacttgt gccacggttt10321 gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtaataat ccccagggag10381 gccatgcgcc acggaagctg tacgcgtggc atattggact agcggttaga ggagacccct10441 cccatcactg ataaaacgca gcaaaagggg gcccgaagcc aggaggaagc tgtactcctg10501 gtggaaggac tagaggttag aggagacccc cccaacacaa aaacagcata ttgacgctgg10561 gaaagaccag agatcctgct gtctctgcaa catcaatcca ggcacagagc gccgcaagat10621 ggattggtgt tgttgatcca acaggttct

APPENDIX 3Sequence of recombinant dengue type 2 chimeric virus strain rDEN2/4Δ30LOCUS       Submission pendingDEFINITION  Dengue virus type 2 recombinant clone rDEN2/4030, completesequence. ACCESSION   Submission pending VERSION KEYWORDS    .SOURCE      Dengue virus type 2 NGC.   ORGANISM  Dengue virus type 2            Viruses; ssRNA positive-strand viruses, no DNA stage;Flaviviridae;             Flavivirus; Dengue virus group.REFERENCE   1 (bases 1 to 10616)   AUTHORS.   TITLE  JOURNAL   Unpublished FEATURES             Location/Qualifiers     source          1 . . . 10616                      /organism =“Dengue virus type 2”                      /clone = “rDEN2/4Δ30”     mat_peptide     97 . . . 438                      /product =“anchored capsid (anchC) protein”      mat_peptide     97 . . . 396                     /product = “virion capsid (virC) protein”     CDS             97 . . . 10263                      /codon_start =1                      /product = “polyprotein precursor” /translation =MNNQRKKARNTPFNMLKRERNRVSTVQQLTKRFSLGMLQGRGPLKLFMALVAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRRTAGMIIMLIPTVMAFHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTLMAMDLGELCEDTITYKCPLLRQNEPEDIDCWCNSTSTWVTYGTCTTTGEHRREKRSVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWILRHPGFTIMAAILAYTIGTTHFQRALIFILLTAVAPSMTMRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTCKKNMEGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSITEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFETTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNSRNTSMAMTCIAVGGITLFLGFTVQADMGCVASWSGKELKCGSGIFVVDNVHTWTEQYKFQPESPARLASAILNAHKDGVCGIRSTTRLENVMWKQITNELNYVLWEGGHDLTVVAGDVKGVLTKGKRALTPPVSDLKYSWKTWGKAKIFTPEARNSTFLIDGPDTSECPNERRAWNSLEVEDYGFGMFTTNIWMKFREGSSEVCDHRLMSAAIEDQKAVHADMGYWIESSKNQTWQIEKASLIEVKTCLWPKTHTLWSNGVLESQMLIPKSYAGPFSQHNYRQGYATQTVGPWHLGKLEIDFGECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCRSCTMPPLRFLGEDGCWYGMEIRPLSEKEENMVKSQVTAGQGTSETFSMGLLCLTLFVEECLRRRVTRKHMILVVVITLCAIILGGLTWMDLLRALIMLGDTMSGRIGGQIHLAIMAVFKMSPGYVLGVFLRKLTSRETALMVIGMAMTTVLSIPHDLMELIDGISLGLILLKIVTQFDNTQVGTLALSLTFIRSTMPLVMAWRTIMAVLFVVTLIPLCRTSCLQKQSHWVEITALILGAQALPVYLMTLMKGASRRSWPLNEGIMAVGLVSLLGSALLKNDVPLAGPMVAGGLLLAAYVMSGSSADLSLEKAANVQWDEMADITGSSPIVEVKQDEDGSFSIRDVEETNMITLLVKLALITVSGLYPLAIPVTMTLWYMWQVKTQRSGALWDVPSPAATKKAALSEGVYRIMQRGLFGKTQVGVGIHMEGVFHTMWHVTRGSVICHETGRLEPSWADVRNDMISYGGGWRLGDKWDKEEDVQVLAIEPGKNPKHVQTKPGLFKTLTGEIGAVTLDFKPGTSGSPIINRKGKVIGLYGNGVVTKSGDYVSAITQAERIGEPDYEVDEDIFRKKRLTIMDLHPGAGKTKRILPSIVREALKRRLRTLILAPTRVVAAEMEEALRGLPIRYQTPAVKSEHTGREIVDLMCHATFTTRLLSSTRVPNYNLIVMDEAHFTDPSSVAARGYISTRVEMGEAAAIFMTATPPGATDPFPQSNSPIEDIEREIPERSWNTGFDWITDYQGKTVWFVPSIKAGNDIANCLRKSGKKVIQLSRKTFDTEYPKTKLTDWDFVVTTDISEMGANFRAGRVIDPRRCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYVFSGDPLKNDEDHAHWTEAKMLLDNIYTPEGIIPTLFGPEREKTQAIDGEFRLRGEQRKTFVELMRRGDLPVWLSYKVASAGISYEDREWCFTGERNNQILEENMEVEIWTREGEKKKLRPRWLDARVYADPMALKDFKEFASGRKSITLDILTEIASLPTYLSSRAKLALDNIVMLHTTERGGRAYQHALNELPESLETLMLVALLGAMTAGIFLFFMQGKGIGKLSMGLITIAVASGLLWVAEIQPQWIAASIILEFFLMVLLIPEPEKQRTPQDNQLIYVILTILTIIGLIAANEMGLIEKTKTDFGFYQVKTETTILDVDLRPASAWTLYAVATTILTPMLRHTIENTSANLSLAAIANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVNPTTLTASLVMLLVHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGITVIDLEPISYDPKFEKQLGQVMLLVLCAGQLLLMRTTWAFCEVLTLATGPILTLWEGNPGRFWNTTIAVSTANIFRGSYLAGAGLAFSLIKNAQTPRRGTGTTGETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGSKIKHAVSRGSSKIRWIVERGMVKPKGKVVDLGCGRGGWSYYMATLKNVTEVKGYTKGGPGHEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTIEEGRTLRVLKMVEPWLSSKPEFCIKVLNPYMPTVIEELEKLQRKHGGNLVRCPLSRNSTHEMYWVSGASGNIVSSVNTTSKMLLNRFTTRHRKPTYEKDVDLGAGTRSVSTETEKPDMTIIGRRLQRLQEEHKETWHYDQENPYRTWAYHGSYEAPSTGSASSMVNGVVKLLTKPWDVIPMVTQLAMTDTTPFGQQRVFKEKVDTRTPQPKPGTRMVMTTTANWLWALLGKKKNPRLCTREEFISKVRSNAAIGAVFQEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMGKREKKLGEFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSWSGVEGEGLHRLGYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQMAPHHKILAKAIFKLTYQNKVVKVLRPTPRGAVMDIISRKDQRGSGQVGTYGLNTFTNMEVQLIRQMEAEGVITQDDMQNPKGLKERVEKWLKECGVDRLKRMAISGDDCVVKPLDERFGTSLLFLNDMGKVRKDIPQWEPSKGWKNWQEVPFCSHHFHKIFMKDGRSLVVPCRNQDELIGRARISQGAGWSLRETACLGKAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWSIHAHHQWMTTEDMLKVWNRVWIEDNPNMTDKTPVHSWEDIPYLGKREDLWCGSLIGLSSRATWAKNIHTAITQVRNLIGKEEYVDYMPVMKRYSAPSESEGVL”     mat_peptide     439 . . . 936                      /product =“membrane precursor (prM) protein”      mat_peptide     712 . . . 936                     /product = “membrane (M) protein”     mat_peptide     937 . . . 2421                      /product =“envelope (E) protein”      mat_peptide     2422 . . . 3477                     /product = “NS1 protein”     mat_peptide     3478 . . . 4131                      /product =“NS2A protein”      mat_peptide     4132 . . . 4521                     /product = “NS2B protein”     mat_peptide     4522 . . . 6375                      /product =“NS3 protein”      mat_peptide     6376 . . . 6756                     /product = “NS4A protein”     mat_peptide     6757 . . . 6825                      /product =“2K protein”      mat_peptide     6826 . . . 7560                     /product = “NS4B protein”     mat_peptide     7561 . . . 10260                      /product =“NS5 protein” rDEN2/4Δ30 sequence    1 agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg   51 cttaacacag ttctaacagt ttgtttgaat agagagcaga tctctgatga  101 ataaccaacg aaaaaaggcg agaaatacgc ctttcaatat gctgaaacgc  151 gagagaaacc gcgtgtcgac tgtacaacag ctgacaaaga gattctcact  201 tggaatgctg cagggacgag gaccattaaa actgttcatg gccctggtgg  251 cgttccttcg tttcctaaca atcccaccaa cagcagggat actgaagaga  301 tggggaacaa ttaaaaaatc aaaagccatt aatgttttga gagggttcag  351 gaaagagatt ggaaggatgc tgaacatctt gaacaggaga cgcagaactg  401 caggcatgat cattatgctg attccaacag tgatggcgtt ccatttaacc  451 acacgtaacg gagaaccaca catgatcgtc agtagacaag agaaagggaa  501 aagtcttctg tttaaaacag aggatggtgt gaacatgtgt accctcatgg  551 ccatggacct tggtgaattg tgtgaagata caatcacgta caagtgtcct  601 cttctcaggc agaatgaacc agaagacata gattgttggt gcaactctac  651 gtccacatgg gtaacttatg ggacgtgtac caccacagga gaacacagaa  701 gagaaaaaag atcagtggca ctcgttccac atgtgggaat gggactggag  751 acacgaactg aaacatggat gtcatcagaa ggggcctgga aacatgccca  801 gagaattgaa acttggatct tgagacatcc aggctttacc ataatggcag  851 caatcctggc atacaccata ggaacgacac atttccaaag agccctgatt  901 ttcatcttac tgacagctgt cgctccttca atgacaatgc gttgcatagg  951 aatatcaaat agagactttg tagaaggggt ttcaggagga agctgggttg 1001 acatagtctt agaacatgga agctgtgtga cgacgatggc aaaaaacaaa 1051 ccaacattgg attttgaact gataaaaaca gaagccaaac aacctgccac 1101 tctaaggaag tactgtatag aggcaaagct gaccaacaca acaacagaat 1151 ctcgctgccc aacacaagga gaacctagcc taaatgaaga gcaggacaaa 1201 aggttcgtct gcaaacactc catggtggac agaggatggg gaaatggatg 1251 tggattattt ggaaaaggag gcattgtgac ctgtgctatg ttcacatgca 1301 aaaagaacat ggaaggaaaa gtcgtgcaac cagaaaactt ggaatacacc 1351 attgtgataa cacctcactc aggggaagag catgcagtcg gaaatgacac 1401 aggaaaacat ggcaaggaaa tcaaaataac accacagagt tccatcacag 1451 aagcagagtt gacaggctat ggcactgtca cgatggagtg ctctccgaga 1501 acgggcctcg acttcaatga gatggtgttg ctgcaaatgg aaaataaagc 1551 ttggctggtg cacaggcaat ggttcctaga cctgccgttg ccatggctgc 1601 ccggagcgga cacacaagga tcaaattgga tacagaaaga gacattggtc 1651 actttcaaaa atccccatgc gaagaaacag gatgttgttg ttttgggatc 1701 ccaagaaggg gccatgcaca cagcactcac aggggccaca gaaatccaga 1751 tgtcatcagg aaacttactg ttcacaggac atctcaagtg caggctgagg 1801 atggacaaac tacagctcaa aggaatgtca tactctatgt gcacaggaaa 1851 gtttaaagtt gtgaaggaaa tagcagaaac acaacatgga acaatagtta 1901 tcagagtaca atatgaaggg gacggttctc catgtaagat cccttttgag 1951 ataatggatt tggaaaaaag acatgtttta ggtcgcctga ttacagtcaa 2001 cccaatcgta acagaaaaag atagcccagt caacatagaa gcagaacctc 2051 cattcggaga cagctacatc atcataggag tagagccggg acaattgaag 2101 ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac 2151 aatgagggga gcgaagagaa tggccatttt aggtgacaca gcttgggatt 2201 ttggatccct gggaggagtg tttacatcta taggaaaggc tctccaccaa 2251 gttttcggag caatctatgg ggctgccttc agtggggtct catggactat 2301 gaaaatcctc ataggagtca ttatcacatg gataggaatg aactcgagga 2351 acacttcaat ggctatgacg tgcatagctg ttggaggaat cactctgttt 2401 ctgggcttca cagttcaagc agacatgggt tgtgtggcgt catggagtgg 2451 gaaagaattg aagtgtggaa gcggaatttt tgtggttgac aacgtgcaca 2501 cttggacaga acagtacaaa tttcaaccag agtccccagc gagactagcg 2551 tctgcaatat taaatgccca caaagatggg gtctgtggaa ttagatcaac 2601 cacgaggctg gaaaatgtca tgtggaagca aataaccaac gagctaaact 2651 atgttctctg ggaaggagga catgacctca ctgtagtggc tggggatgtg 2701 aagggggtgt tgaccaaagg caagagagca ctcacacccc cagtgagtga 2751 tctgaaatat tcatggaaga catggggaaa agcaaaaatc ttcaccccag 2801 aagcaagaaa tagcacattt ttaatagacg gaccagacac ctctgaatgc 2851 cccaatgaac gaagagcatg gaactctctt gaggtggaag actatggatt 2901 tggcatgttc acgaccaaca tatggatgaa attccgagaa ggaagttcag 2951 aagtgtgtga ccacaggtta atgtcagctg caattaaaga tcagaaagct 3001 gtgcatgctg acatgggtta ttggatagag agctcaaaaa accagacctg 3051 gcagatagag aaagcatctc ttattgaagt gaaaacatgt ctgtggccca 3101 agacccacac actgtggagc aatggagtgc tggaaagcca gatgctcatt 3151 ccaaaatcat atgcgggccc tttttcacag cacaattacc gccagggcta 3201 tgccacgcaa accgtgggcc catggcactt aggcaaatta gagatagact 3251 ttggagaatg ccccggaaca acagtcacaa ttcaggagga ttgtgaccat 3301 agaggcccat ctttgaggac caccactgca tctggaaaac tagtcacgca 3351 atggtgctgc cgctcctgca cgatgcctcc cttaaggttc ttgggagaag 3401 atgggtgctg gtatgggatg gagattaggc ccttgagtga aaaagaagag 3451 aacatggtca aatcacaggt gacggccgga cagggcacat cagaaacttt 3501 ttctatgggt ctgttgtgcc tgaccttgtt tgtggaagaa tgcttgagga 3551 gaagagtcac taggaaacac atgatattag ttgtggtgat cactctttgt 3601 gctatcatcc tgggaggcct cacatggatg gacttactac gagccctcat 3651 catgttgggg gacactatgt ctggtagaat aggaggacag atccacctag 3701 ccatcatggc agtgttcaag atgtcaccag gatacgtgct gggtgtgttt 3751 ttaaggaaac tcacttcaag agagacagca ctaatggtaa taggaatggc 3801 catgacaacg gtgctttcaa ttccacatga ccttatggaa ctcattgatg 3851 gaatatcact gggactaatt ttgctaaaaa tagtaacaca gtttgacaac 3901 acccaagtgg gaaccttagc tctttccttg actttcataa gatcaacaat 3951 gccattggtc atggcttgga ggaccattat ggctgtgttg tttgtggtca 4001 cactcattcc tttgtgcagg acaagctgtc ttcaaaaaca gtctcattgg 4051 gtagaaataa cagcactcat cctaggagcc caagctctgc cagtgtacct 4101 aatgactctt atgaaaggag cctcaagaag atcttggcct cttaacgagg 4151 gcataatggc tgtgggtttg gttagtctct taggaagcgc tcttttaaag 4201 aatgatgtcc ctttagctgg cccaatggtg gcaggaggct tacttctggc 4251 ggcttacgtg atgagtggta gctcagcaga tctgtcacta gagaaggccg 4301 ccaacgtgca gtgggatgaa atggcagaca taacaggctc aagcccaatc 4351 atagaagtga agcaggatga agatggctct ttctccatac gggacgtcga 4401 ggaaaccaat atgataaccc ttttggtgaa actggcactg ataacagtgt 4451 caggtctcta ccccttggca attccagtca caatgacctt atggtacatg 4501 tggcaagtga aaacacaaag atcaggagcc ctgtgggacg tcccctcacc 4551 cgctgccact aaaaaagccg cactgtctga aggagtgtac aggatcatgc 4601 aaagagggtt attcgggaaa actcaggttg gagtagggat acacatggaa 4651 ggtgtatttc acacaatgtg gcatgtaaca agaggatcag tgatctgcca 4701 cgagactggg agattggagc catcttgggc tgacgtcagg aatgacatga 4751 tatcatacgg tgggggatgg aggcttggag acaaatggga caaagaagaa 4801 gacgttcagg tcctcgccat agaaccagga aaaaatccta aacatgtcca 4851 aacgaaacct ggccttttca agaccctaac tggagaaatt ggagcagtaa 4901 cattagattt caaacccgga acgtctggtt ctcccatcat caacaggaaa 4951 ggaaaagtca tcggactcta tggaaatgga gtagttacca aatcaggtga 5001 ttacgtcagt gccataacgc aagccgaaag aattggagag ccagattatg 5051 aagtggatga ggacattttt cgaaagaaaa gattaactat aatggactta 5101 caccccggag ctggaaagac aaaaagaatt cttccatcaa tagtgagaga 5151 agccttaaaa aggaggctac gaactttgat tttagctccc acgagagtgg 5201 tggcggccga gatggaagag gccctacgtg gactgccaat ccgttatcag 5251 accccagctg tgaaatcaga acacacagga agagagattg tagacctcat 5301 gtgtcatgca accttcacaa caagactttt gtcatcaacc agggttccaa 5351 attacaacct tatagtgatg gatgaagcac atttcaccga tccttctagt 5401 gtcgcggcta gaggatacat ctcgaccagg gtggaaatgg gagaggcagc 5451 agccatcttc atgaccgcaa cccctcccgg agcgacagat ccctttcccc 5501 agagcaacag cccaatagaa gacatcgaga gggaaattcc ggaaaggtca 5551 tggaacacag ggttcgactg gataacagac taccaaggga aaactgtgtg 5601 gtttgttccc agcataaaag ctggaaatga cattgcaaat tgtttgagaa 5651 agtcgggaaa gaaagttatc cagttgagta ggaaaacctt tgatacagag 5701 tatccaaaaa cgaaactcac ggactgggac tttgtggtca ctacagacat 5751 atctgaaatg ggggccaatt ttagagccgg gagagtgata gaccctagaa 5801 gatgcctcaa gccagttatc ctaccagatg ggccagagag agtcatttta 5851 gcaggtccta ttccagtgac tccagcaagc gctgctcaga gaagagggcg 5901 aataggaagg aacccagcac aagaagacga ccaatacgtt ttctccggag 5951 acccactaaa aaatgatgaa gatcatgccc actggacaga agcaaagatg 6001 ctgcttgaca atatctacac cccagaaggg atcattccaa cattgtttgg 6051 tccggaaagg gaaaaaaccc aagccattga tggagagttt cgcctcagag 6101 gggaacaaag gaagactttt gtggaattaa tgaggagagg agaccttccg 6151 gtgtggctga gctataaggt agcttctgct ggcatttctt acaaagatcg 6201 ggaatggtgc ttcacagggg aaagaaataa ccaaatttta gaagaaaaca 6251 tggaggttga aatttggact agagagggag aaaagaaaaa gctaaggcca 6301 agatggttag atgcacgtgt atacgctgac cccatggctt tgaaggattt 6351 caaggagttt gccagtggaa ggaagagtat aactctcgac atcctaacag 6401 agattgccag tttgccaact tacctttcct ctagggccaa gctcgccctt 6451 gataacatag tcatgctcca cacaacagaa agaggaggga gggcctatca 6501 acacgccctg aacgaacttc cggagtcact ggaaacactc atgcttgtag 6551 ctttactagg tgctatgaca gcaggcatct tcctgttttt catgcaaggg 6601 aaaggaatag ggaaattgtc aatgggtttg ataaccattg cggtggctag 6651 tggcttgctc tgggtagcag aaattcaacc ccagtggata gcggcctcaa 6701 tcatactaga gttttttctc atggtactgt tgataccgga accagaaaaa 6751 caaaggaccc cacaagacaa tcaattgatc tacgtcatat tgaccattct 6801 caccatcatt ggtctaatag cagccaacga gatggggctg attgaaaaaa 6851 caaaaacgga ttttgggttt taccaggtaa aaacagaaac caccatcctc 6901 gatgtggact tgagaccagc ttcagcatgg acgctctatg cagtagccac 6951 cacaattctg actcccatgc tgagacacac catagaaaac acgtcggcca 7001 acctatctct agcagccatt gccaaccagg cagccgtcct aatggggctt 7051 ggaaaaggat ggccgctcca cagaatggac ctcggtgtgc cgctgttagc 7101 aatgggatgc tattctcaag tgaacccaac aaccttgaca gcatccttag 7151 tcatgctttt agtccattat gcaataatag gcccaggatt gcaggcaaaa 7201 gccacaagag aggcccagaa aaggacagct gctgggatca tgaaaaatcc 7251 cacagtggac gggataacag taatagatct agaaccaata tcctatgacc 7301 caaaatttga aaagcaatta gggcaggtca tgctactagt cttgtgtgct 7351 ggacaactac tcttgatgag aacaacatgg gctttctgtg aagtcttgac 7401 tttggccaca ggaccaatct tgaccttgtg ggagggcaac ccgggaaggt 7451 tttggaacac gaccatagcc gtatccaccg ccaacatttt caggggaagt 7501 tacttggcgg gagctggact ggctttttca ctcataaaga atgcacaaac 7551 ccctaggagg ggaactggga ccacaggaga gacactggga gagaagtgga 7601 agagacagct aaactcatta gacagaaaag agtttgaaga gtataaaaga 7651 agtggaatac tagaagtgga caggactgaa gccaagtctg ccctgaaaga 7701 tgggtctaaa atcaagcatg cagtatcaag agggtccagt aagatcagat 7751 ggattgttga gagagggatg gtaaagccaa aagggaaagt tgtagatctt 7801 ggctgtggga gaggaggatg gtcttattac atggcgacac tcaagaacgt 7851 gactgaagtg aaagggtata caaaaggagg tccaggacat gaagaaccga 7901 ttcccatggc tacttatggt tggaatttgg tcaaactcca ttcaggggtt 7951 gacgtgttct acaaacccac agagcaagtg gacaccctgc tctgtgatat 8001 tggggagtca tcttctaatc caacaataga ggaaggaaga acattaagag 8051 ttttgaagat ggtggagcca tggctctctt caaaacctga attctgcatc 8101 aaagtcctta acccctacat gccaacagtc atagaagagc tggagaaact 8151 gcagagaaaa catggtggga accttgtcag atgcccgctg tccaggaact 8201 ccacccatga gatgtattgg gtgtcaggag cgtcgggaaa cattgtgagc 8251 tctgtgaaca caacatcaaa gatgttgttg aacaggttca caacaaggca 8301 taggaaaccc acttatgaga aggacgtaga tcttggggca ggaacgagaa 8351 gtgtctccac tgaaacagaa aaaccagaca tgacaatcat tgggagaagg 8401 cttcagcgat tgcaagaaga gcacaaagaa acctggcatt atgatcagga 8451 aaacccatac agaacctggg cgtatcatgg aagctatgaa gctccttcga 8501 caggctctgc atcctccatg gtgaacgggg tggtaaaact gctaacaaaa 8551 ccctgggatg tgattccaat ggtgactcag ttagccatga cagatacaac 8601 cccttttggg caacaaagag tgttcaaaga gaaggtggat accagaacac 8651 cacaaccaaa acccggtaca cgaatggtta tgaccacgac agccaattgg 8701 ctgtgggccc tccttggaaa gaagaaaaat cccagactgt gcacaaggga 8751 agagttcatc tcaaaagtta gatcaaacgc agccataggc gcagtctttc 8801 aggaagaaca gggatggaca tcagccagtg aagctgtgaa tgacagccgg 8851 ttttgggaac tggttgacaa agaaagggcc ctacaccagg aagggaaatg 8901 tgaatcgtgt gtctataaca tgatgggaaa acgtgagaaa aagttaggag 8951 agtttggcag agccaaggga agccgagcaa tctggtacat gtggctggga 9001 gcgcggtttc tggaatttga agccctgggt tttttgaatg aagatcactg 9051 gtttggcaga gaaaattcat ggagtggagt ggaaggggaa ggtctgcaca 9101 gattgggata tatcctggag gagatagaca agaaggatgg agacctaatg 9151 tatgctgatg acacagcagg ctgggacaca agaatcactg aggatgacct 9201 tcaaaatgag gaactgatca cggaacagat ggctccccac cacaagatcc 9251 tagccaaagc cattttcaaa ctaacctatc aaaacaaagt ggtgaaagtc 9301 ctcagaccca caccgcgggg agcggtgatg gatatcatat ccaggaaaga 9351 ccaaagaggt agtggacaag ttggaacata tggtttgaac acattcacca 9401 acatggaagt tcaactcatc cgccaaatgg aagctgaagg agtcatcaca 9451 caagatgaca tgcagaaccc aaaagggttg aaagaaagag ttgagaaatg 9501 gctgaaagag tgtggtgtcg acaggttaaa gaggatggca atcagtggag 9551 acgattgcgt ggtgaagccc ctagatgaga ggtttggcac ttccctcctc 9601 ttcttgaacg acatgggaaa ggtgaggaaa gacattccgc agtgggaacc 9651 atctaaggga tggaaaaact ggcaagaggt tcctttttgc tcccaccact 9701 ttcacaagat ctttatgaag gatggccgct cactagttgt tccatgtaga 9751 aaccaggatg aactgatagg gagagccaga atctcgcagg gagctggatg 9801 gagcttaaga gaaacagcct gcctgggcaa agcttacgcc cagatgtggt 9851 cgcttatgta cttccacaga agggatctgc gtttagcctc catggccata 9901 tgctcagcag ttccaacgga atggtttcca acaagcagaa caacatggtc 9951 aatccacgct catcaccagt ggatgaccac tgaagatatg ctcaaagtgt10001 ggaacagagt gtggatagaa gacaacccta atatgactga caagactcca10051 gtccattcgt gggaagatat accttaccta gggaaaagag aggatttgtg10101 gtgtggatcc ctgattggac tttcttccag agccacctgg gcgaagaaca10151 ttcacacggc cataacccag gtcaggaacc tgatcggaaa agaggaatac10201 gtggattaca tgccagtaat gaaaagatac agtgctcctt cagagagtga10251 aggagttctg taattaccaa caacaaacac caaaggctat tgaagtcagg10301 ccacttgtgc cacggtttga gcaaaccgtg ctgcctgtag ctccgccaat10351 aatgggaggc gtaataatcc ccagggaggc catgcgccac ggaagctgta10401 cgcgtggcat attggactag cggttagagg agacccctcc catcactgac10451 aaaacgcagc aaaagggggc ccaagactag aggttagagg agaccccccc10501 aacacaaaaa cagcatattg acgctgggaa agaccagaga tcctgctgtc10551 tctgcaacat caatccaggc acagagcgcc gcaagatgga ttggtgttgt10601 tgatccaaca ggttct

APPENDIX 4 Alignment of dengue virus polyproteins DEN4 1 MNQRKKVVRPPFNMLKRERNRVSTPQGLVKRFSTGLFSGKGPLRMVLAF 49 DEN1-WP 1MNNQRKKTGRPSFNMLKRARNRVSTVSQLAKRFSKGLLSGQGPMKLVMAF 50 DEN2-NGC 1MNNQRKKARNTPFNMLKRERNRVSTVQQLTKRFSLGMLQGRGPLKLFMAL 50 DEN3-H87 1MNNQRKKTGKPSINMLKRVRNRVSTGSQLAKRFSRGLLNGQGPMKLVMAF 50  *****      ***** ******   * **** *.  *.**... .*  DEN4 50ITFLRVLSIPPTAGILKRWGQLKKNKAIKILIGFRKEIGRMLNILNGRKR 99 DEN1-WP 51IAFLRFLAIPPTAGILARWGSFKKNGAIKVLRGFKKEISNMLNIMNRRKR 100 DEN2-NGC 51VAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRR 100 DEN3-H87 51IAFLRFLAIPPTAGVLARWGTFKKSGAIKVLKGFKKEISNMLSIINKRKK 100..*** *.******.* ***  **  ** .* **.***  ** *.* *..  DEN4 100STITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTL 149 DEN1-WP 101SVTMLLMLLPTALAFHLTTRGGEPHMIVSKQERGKSLLFKTSAGVNMCTL 150 DEN2-NGC 101TAGMIIMLIPTVMAFHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTL 150 DEN3-H87 101TSLCLMMMLPATLAFHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTL 150.   .. ..*. .** *..* *** *** . *.*. *****  *.* ***  DEN4 150IAMDLGEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGER 199 DEN1-WP 151IAMDLGELCEDTMTYKCPRITETEPDDVDCWCNATETWVTYGTCSQTGEH 200 DEN2-NGC 151MAMDLGELCEDTITYKCPFLRQNEPEDIDCWCNSTSTWVTYGTCTTTGEH 200 DEN3-H87 151IAMDLGEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEH 200.******.*.**.***** .   **.*.***** * *** ****  .**.  DEN4 200RREKRSVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILRNPGFALL 249 DEN1-WP 201RRDKRSVALAPHVGLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVI 250 DEN2-NGC 201RREKRSVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWILRHPGFTIM 250 DEN3-H87 201RRDKRSVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTIL 250**.****** ** *.**.**..****.****.. ...*.* **.***...  DEN4 250AGFMAYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGGAW 299 DEN1-WP 251ALFLAHAIGTSITQKGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATW 300 DEN2-NGC 251AAILAYTIGTTHFQRALIFILLTAVAPSMTMRCIGISNRDFVEGVSGGSW 300 DEN3-H87 251ALFLAHYIGTSLTQKVVIFILLMLVTPSMTMRCVGVGNRDFVEGLSGATW 300*  .*  ** .  *. . *.*.  *.**  ***.*. *******.** .*  DEN4 300VDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASISNITT 349 DEN1-WP 301VDVVLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTT 350 DEN2-NGC 301VDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTT 350 DEN3-H87 301VDVVLEHGGCVTTMAKNKPTLDIELQKTEATQLATLRKLCIEGKITNITT 350**.***** ******. ***** ** **     * **  ***  ..* **  DEN4 350ATRCPTQGEPYLKEEQDQQYICRRDVVDRGWGNGCGLFGKGGVVTCAKFS 399 DEN1-WP 351DSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCAKFK 400 DEN2-NGC 351DSRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFT 400 DEN3-H87 351DSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQ 400 .*******  * ****  ..*..  *************** ..*** *  DEN4 400CSGKITGNLVQIENLEYTVVVTVHNGDTHAVGNDTSNHGVTAMITPRSPS 449 DEN1-WP 401CVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTTATITPQAPT 450 DEN2-NGC 401CKKNMKGKVVQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSI 450 DEN3-H87 401CLESIEGKVVQHENLKYTVIITVHTGDQHQVGNET--QGVTAEITSQAST 448*   . * .** *** *....* * *. * ***.*  .*    ** ..  DEN4 450VEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPW 499 DEN1-WP 451SEIQLTDYGALTLDCSPRTGLDFNEMVLLTMEKKSWLVHKQWFLDLPLPW 500 DEN2-NGC 451TEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPW 500 DEN3-H87 449AEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPW 498 *  *  ** . ..* **.*.*****.*. *  *.*.**.*** ******  DEN4 500TAGADTSEVHWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEV 549 DEN1-WP 501TSGASTSQETWNRQDLLVTFKTAHAKKQEVVVLGSQEGAMHTALTGATEI 550 DEN2-NGC 501LPGADTQGSNWIQKETLVTFKNPHAKKQDVVVLGSQEGAMHTALTGATEI 550 DEN3-H87 499TSGATTKTPTWNRKELLVTFKNAHAKKQEVVVLGSQEGAMHTALTGATEI 548  ** *    *  .. .****  ***.*.* **********.**.****.  DEN4 550DSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTT 599 DEN1-WP 551QTSGTTTIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTV 600 DEN2-NGC 551QMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKVVKEIAETQHGTI 600 DEN3-H87 549QTSGGTSIFAGHLKCRLKMDKLKLKGMSYAMCLNTFVLKKEVSETQHGTI 598     . .*.*****...*.** .***** ** . * . **..******  DEN4 600VVKVKYEGAGAPCKVPIEIRDVNKEKVVGRIISSTPLAENTNSVTNIELE 649 DEN1-WP 601LVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAE 650 DEN2-NGC 601VIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAE 650 DEN3-H87 599LIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPVVTKKEEPVNIEAE 648...* * *  .***.*    *       **.*. .*.        *** *  DEN4 650PPFGDSYIVIGVGNSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAW 699 DEN1-WP 651PPFGESYIVVGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAW 700 DEN2-NGC 651PPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAILGDTAW 700 DEN3-H87 649PPFGESNIVIGIGDKALKINWYRKGSSIGKMFEATARGARRMAILGDTAW 698****.* *..*     * . *..******.* *.* ***.******.***  DEN4 700DFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLVLWIGTNS 749 DEN1-WP 701DFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNS 750 DEN2-NGC 701DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNS 750 DEN3-H87 699DFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWIMKIGIGVLLTWIGLNS 748****.**. .*.** .**.**. *   * **** ..* ** .. *.* **  DEN4 750RNTSMAMTCIAVGGITLFLGFTVQADMGCVASWSGKELKCGSGIFVVDNV 799 DEN1-WP 751RSTSLSMTCIAVGMVTLYLGVMVQADSGCVINWKGRELKCGSGIFVTNEV 800 DEN2-NGC 751RSTSLSVSLVLVGVVTLYLGVMVQADSGCVVSWKNKELKCGSGIFITDNV 800 DEN3-H87 749KNTSMSFSCIAIGIITLYLGVVVQADMGCVINWKGKELKCGSGIFVTNEV 798. **.. . . .* .**.**  **** ***  * ..*********.   *  DEN4 800HTWTEQYKFQPESPARLASAILNAHKDGVCGIRSTTRLENVMWKQITNEL 849 DEN1-WP 801HTWTEQYKFQADSPKRLSAAIGKAWEEGVCGIRSATRLENIMWKQISNEL 850 DEN2-NGC 801HTWTEQYKFQPESPSKLASAIQKAHEEGICGIRSVTRLENLMWKQITPEL 850 DEN3-H87 799HTWTEQYKFQADSPKRVATAIAGAWENGVCGIRSTTRMENLLWKQIANEL 848********** .** ....**  *   *.***** **.**..****. **  DEN4 850NYVLWEGGHDLTVVAGDVKGVLTKGKRALTPPVSDLKYSWKTWGKAKIFT 899 DEN1-WP 851NHILLENDMKFTVVVGDVSGILAQGKKMIRPQPMEHKYSWKSWGKAKIIG 900 DEN2-NGC 851NHILSENEVKLTIMTGDIKGIMQAGKRSLQPQPTELKYSWKTWGKAKMLS 900 DEN3-H87 849NYILWENDIKLTVVVGDITGVLEQGKRTLTPQPMELKYSWKTWGLAKIVT 898* .* *.    *.. **. *..  **. . *   . *****.** **.  DEN4 900PEARNSTFLIDGPDTSECPNERRAWNSLEVEDYGFGMFTTNIWMKFREGS 949 DEN1-WP 901ADVQNTTFIIDGPNTPECPDNQRAWNIWEVEDYGFGIFTTNIWLKLRDSY 950 DEN2-NGC 901TESHNQTFLIDGPETAECPNTNRAWNSLEVEDYGFGVETTNIWLKLREKQ 950 DEN3-H87 899AETQNSSFIIDGPSTPECPSASRAWNVWEVEDYGFGVFTTNIWLKLREVY 948 . .* .*.**** * ***   ****  ********.******.* *.  DEN4 950SEVCDHRLMSAAIKDQKAVHADMGYWIESSKNQTWQIEKASLIEVKTCLW 999 DEN1-WP 951TQVCDHRLMSAAIKDSKAVHADMGYWIESEKNETWKLARASFIEVKTCIW 1000 DEN2-NGC 951DVFCDSKLMSAAIKDNRAVHADMGYWIESALNDTWKIEKASFIEVKSCHW 1000 DEN3-H87 949TQLCDHRLMSAAVKDERAVHADMGYWIESQKNGSWKLEKASLIEVKTCTW 998   ** .*****.** .************  * .*.. .** ****.* *  DEN4 1000PKTHTLWSNGVLESQMLIPKSYAGPFSQHNYRQGYATQTVGPWHLGKLEI 1049 DEN1-WP 1001PKSHTLWSNGVLESEMIIPKIYGGPISQHNYRPGYFTQTAGPWHLGKLEL 1050 DEN2-NGC 1001PKSHTLWSNGVLESEMIIPKNFAGPVSQHNYRPGYHTQTAGPWHLGKLEM 1050 DEN3-H87 999PKSHTLWSNGVLESDMIIPKSLAGPISQHNHRPGYHTQTAGPWHLGKLEL 1048**.*********** *.***   ** **** * ** *** *********.  DEN4 1050DFGECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCRSCTMPPLRFLG 1099 DEN1-WP 1051DFDLCEGTTVVVDEHCGNRGPSLRTTTVTGKTIHEWCCRSCTLPPLRFKG 1100 DEN2-NGC 1051DFDFCEGTTVVVTEDCGNRGPSLRTTTASGKLITEWCCRSCTLPPLRYRG 1100 DEN3-H87 1049DFNYCEGTTVVISENCGTRGPSLRTTTVSGKLIHEWCCRSCTLPPLRYMG 1098**  * **** . * *  ********* .** . .*******.****. *  DEN4 1100EDGCWYGMEIRPLSEKEENMVKSQVTAGQGTSETFSMGLLCLTLFVEECL 1149 DEN1-WP 1101EDGCWYGMEIRPVKEKEENLVKSMVSAGSGEVDSFSLGLLCISIMIEEVM 1150 DEN2-NGC 1101EDGCWYGMEIRPLKEKEENLVNSLVTAGHGQIDNFSLGVLGMALFLEEML 1150 DEN3-H87 1099EDGCWYGMEIRPINEKEENMVKSLASAGSGKVDNFTMGVLCLAILFEEVM 1148************. *****.* *  .** *  . *..*.* ...  ** .  DEN4 1150RRRVTRKHMILVVVITLCAIILGGLTWMDLLRALIMLGDTMSGRIG-GQI 1198 DEN1-WP 1151RSRWSRKMLMTGTLAVFLLLTMGQLTWNDLIRLCIMVGANASDKMGMGTT 1200 DEN2-NGC 1151RTRVGTKHAILLVAVSFVTLITGNMSFRDLGRVMVMVGATMTDDIGMGVT 1200 DEN3-H87 1149RGKFGKKHMIAGVLFTFVLLLSGQITWRGMAHTLIMIGSNASDRMGMGVT 1198* .   *  .         .  * ..   . .  .*.* . .  .* *  DEN4 1199HLAIMAVFKMSPGYVLGVFLRKLTSRETALMVIGMAMTTVLSIPHDLMEL 1248 DEN1-WP 1201YLALMATFRMRPMFAVGLLFRRLTSREVLLLTVGLSLVASVELPNSLEEL 1250 DEN2-NGC 1201YLALLAAFKVRPTFAAGLLLRKLTSKELMMTTIGIVLLSQSTIPETILEL 1250 DEN3-H87 1199YLALIATFKIQPFLALGFFLRKLTSRENLLLGVGLAMAATLRLPEDIEQM 1248 **..* *.. *    *   *.***.*  .  .*. . .   .*  . ..  DEN4 1249IDGISLGLILLKIVTQFDNTQVGTLALSLTFIRSTMPLVMAWRTIMAVLF 1298 DEN1-WP 1251GDGLAMGIMMLKLLTDFQSHQLWATLLSLTFVKTTFSLHYAWKTMAMILS 1300 DEN2-NGC 1251TDALALGMMVLKMVRKMEKYQLAVTIMAILCVPNAVILQNAWKVSCTILA 1300 DEN3-H87 1249ANGIALGLMALKLITQFETYQLWTALVSLTCSNTIFTLTVAWRTATLILA 1298   ...*.. **..      *.    ...        *  **.    .*  DEN4 1299VVTLIPLCRTSCLQKQSHWVEITALILGAQALPVYLMTLMKGASRRSWPL 1348 DEN1-WP 1301IVSLFPLCLSTTSQK-TTWLPVLLGSLGCKPLTMFLITENKIWGRKSWPL 1349 DEN2-NGC 1301VVSVSPLFLTSSQQK-ADWIPLALTIKGLNPTAIFLTTLSRTNKKRSWPL 1349 DEN3-H87 1299GISLLPVCQSSSMRK-TDWLPMTVAAMGVPPLPLFIFSLKDTLKRRSWPL 1347 ... *.  ... .* . *. .     *     ... .      ..****  DEN4 1349NEGIMAVGLVSLLGSALLKNDVPLAGPMVAGGLLLAAYVMSGSSADLSLE 1398 DEN1-WP 1350NEGIMAVGIVSILLSSLLKNDVPLAGPLIAGGMIACYVISGSSADLSLE 1399 DEN2-NGC 1350NEAIMAVGMVSILASSLLKNDIPMTGPLVAGGLLTVCYVLTGRSADLELE 1399 DEN3-H87 1348NEGVMAVGLVSILASSLLRNDVPMAGPLVAGGLLIACYVITGTSADLTVE 1397** .****.**.* *.**.**.*..**..***.*  .**..* **** .*  DEN4 1399KAANVQWDEMADITGSSPIIEVKQDEDGSFSIRDVEETNMITLLVKLALI 1448 DEN1-WP 1400KAAEVSWEEEAEHSGASHNILVEVQDDGTMKIKDEERDDTLTILLKATLL 1449 DEN2-NGC 1400RAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLL 1449 DEN3-H87 1398KAADVTWEEEAEQTGVSHNLMITVDDDGTMRIKDDETENILTVLLKTALL 1447.** * *.. *. .* *  . .   .**.  *.  *     .*.*.. *.  DEN4 1449TVSGLYPLAIPVTMTLWYMWQVKTQRSGALWDVPSPAATKKAALSEGVYR 1498 DEN1-WP 1450AISGVYPMSIPATLFVWYFWQKKKQRSGVLWDTPSPPEVERAVLDDGIYR 1499 DEN2-NGC 1450VISGLFPVSIPITAAAWYLWEVKKQRAGVLWDVPSPPPVGKAELEDGAYR 1499 DEN3-H87 1448IVSGIFPYSIPATMLVWHTWQKQTQRSGVLWDVPSPPETQKAELEEGVYR 1497 .**..* .** *   *  *. . **.* *** ***    .* * .* **  DEN4 1499IMQRGLFGKTQVGVGIHMEGVFHTMWHVTRGSVICHETGRLEPSWADVRN 1548 DEN1-WP 1500ILQRGLLGRSQVGVGVFQEGVFHTMWHVTRGAVLMYQGKRLEPSWASVKK 1549 DEN2-NGC 1500IKQKGILGYSQIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEPSWADVKK 1549 DEN3-H87 1498IKQQGIFGKTQVGVGVQKEGVFHTMWHVTRGAVLTHNGKRLEPNWASVKK 1547* *.*. * .*.* *.  ** **********.*.     *.** ** *.  DEN4 1549DMISYGGGWRLGDKWDKEEDVQVLAIEPGKNPKHVQTKPGLFKTLTGEIG 1598 DEN1-WP 1550DLISYGGGWRFQGSWNAGEEVQVIAVEPGKNPKNVQTAPGTFKTPEGEVG 1599 DEN2-NGC 1550DLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIG 1599 DEN3-H87 1548DLISYGGGWRLSAQWQKGEEVQVIAVEPGKNPKNFQTMPGIFQTTTGEIG 1597*.*******.    *   *.***.*.******.  ** ** *.*  * .*  DEN4 1599AVTLDFKPGTSGSPIINRKGKVIGLYGNGVVTKSGDYVSAITQAERIGEP 1648 DEN1-WP 1600AIALDFKPGTSGSPIVNREGKIVGLYGNGVVTTSGTYVSAIAQAKASQEG 1649 DEN2-NGC 1600AVSLDFSPGTSGSPIIDKKGKVVGLYGNGVVTRSGAYVSAIAQTEKSIED 1649 DEN3-H87 1598AIALDFKPGTSGSPIINREGKVVGLYGNGVVTKNGGYVSGIAQTNAEPDG 1647*..*** ********. . **..*********  * *** *.*.    .  DEN4 1649-DYEVDEDIFRKKRLTIMDLHPGAGKTKRILPSIVREALKRRLRTLILAP 1697 DEN1-WP 1650PLPEIEDEVFRKRNLTIMDLHPGSGKTRRYLPAIVREAIRRNVRTLVLAP 1699 DEN2-NGC 1650-NPEIEDDIFRKRKLTIMDLHPGAGKTKRYLPAIVREAIKRGLRTLILAP 1698 DEN3-H87 1648PTPELEEEMFKKRNLTIMDLHPGSGKTRKYLPAIVREAIKRRLRTLILAP 1697   *.....*.*. *********.***.. **.*****..* .***.***  DEN4 1698TRVVAAEMEEALRGLPIRYQTPAVKSEHTGREIVDLMCHATFTTRLLSST 1747 DEN1-WP 1700TRVVASEMAEALKGMPIRYQTTAVKSEHTGKEIVDLMCHATFTMRLLSPV 1749 DEN2-NGC 1699TRVVAAEMEEALRGLPIRYQTPAIRAEHTGREIVDLMCHATFTMRLLSPV 1748 DEN3-H87 1698TRVVAAEMEEAMKGLPIRYQTTATKSEHTGREIVDLMCHATFTMRLLSPV 1747*****.** **..*.****** * ..****.************ ****  DEN4 1748RVPNYNLIVMDEAHFTDPSSVAARGYISTRVEMGEAAAIFMTATPPGATD 1797 DEN1-WP 1750RVPNYNMIIMDEAHFTDPASIAARGYISTRVGMGEAAAIFMTATPPGSVE 1799 DEN2-NGC 1749RVPNYNLIIMDEAHFTDPASIAARGYISTRVEMGEAAGIFMTATPPGSRD 1798 DEN3-H87 1748RVPNYNLIIMDEAHFTDPASIAARGYISTRVGMGEAAAIFMTATPPGTAD 1797******.*.*********.*.********** ***** *********. .  DEN4 1798PFPQSNSPIEDIEREIPERSWNTGFDWITDYQGKTVWFVPSIKAGNDIAN 1847 DEN1-WP 1800AFPQSNAVIQDEERDIPERSWNSGYDWITDFPGKTVWFVPSIKSGNDIAN 1849 DEN2-NGC 1799PFPQSNAPIMDEEREIPERSWSSGHEWVTDFKGKTVWFVPSIKAGNDIAA 1848 DEN3-H87 1798AFPQSNAPIQDEERDIPERSWNSGNEWITDFVGKTVWFVPSIKAGNVIAN 1847 *****. * * **.****** .* .*.**. ***********.** **  DEN4 1848CLRKSGKKVIQLSRKTFDTEYPKTKLTDWDFVVTTDISEMGANFRAGRVI 1897 DEN1-WP 1850CLRKNGKRVVQLSRKTFDTEYQKTKNNDWDYVVTTDISEMGANFRADRVI 1899 DEN2-NGC 1849CLRKNGKKVIQLSRKTFDSEYVKTRTNDWDFVVTTDISEMGANFKAERVI 1898 DEN3-H87 1848CLRKNGKKVIQLSRKTFDTEYQKTKLNDWDFVVTTDISEMGANFIADRVI 1897**** **.*.********.** **. .***.************* * ***  DEN4 1898DPRRCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYV 1947 DEN1-WP 1900DPRRCLKPVILKDGPERVILAGPMPVTVASAAQRRGRIGRNQNKEGDQYI 1949 DEN2-NGC 1899DPRRCMKPVILTDGEERVILAGPMPVTHSSAAQRRGRIGRNPKNENDQYI 1948 DEN3-H87 1898DPRRCLKPVILTDGPERVILAGPMPVTVASAAQRRGRVGRNPQKENDQYI 1947*****.***** ** ********.*** .********.***   * ***.  DEN4 1948FSGDPLKNDEDHAHWTEAKMLLDNIYTPEGIIPTLFGPEREKTQAIDGEF 1997 DEN1-WP 1950YMGQPLNNDEDHAHWTEAKMLLDNINTPEGIIPALFEPEREKSAAIDGEY 1999 DEN2-NGC 1949YMGEPLENDEDCAHWKEAKMLLDNINTPEGIIPSMFEPEREKVDAIDGEY 1998 DEN3-H87 1948FMGQPLNKDEDHAHWTEAKMLLDNINTPEGIIPALFEPEREKSAAIDGEY 1997. * **  *** *** ********* *******..* *****  *****.  DEN4 1998RLRGEQRKTFVELMRRGDLPVWLSYKVASAGISYKDREWCFTGERNNQIL 2047 DEN1-WP 2000RLRGEARKTFVELMRRGDLPVWLSYKVASEGFQYSDRRWCFDGERNNQVL 2049 DEN2-NGC 1999RLRGEARKTFVDLMRRGDLPVWLAYRVAAEGINYADRRWCFDGIKNNQIL 2048 DEN3-H87 1998RLKGESRKTFVELMRRGDLPVWLAHKVASEGIKYTDRKWCFDGERNNQIL 2047**.** *****.***********. .**. *  * ** *** * .***.*  DEN4 2048EENMEVEIWTREGEKKKLRPRWLDARVYADPMALKDFKEFASGRKSITLD 2097 DEN1-WP 2050EENMDVEIWTKEGERKKLRPRWLDARTYSDPLALREFKEFAAGRRSVSGD 2099 DEN2-NGC 2049EENVEVEIWTKEGERKKLKPRWLDARIYSDPLTLKEFKEFAAGRKSLTLN 2098 DEN3-H87 2048EENMDVEIWTKEGEKKKLRPRWLDARTYSDPLALKEFKDFAAGRKSIALD 2097***..*****.***.***.******* *.**..*..**.**.**.*..  DEN4 2098ILTEIASLPTYLSSRAKLALDNIVMLHTTERGGRAYQHALNELPESLETL 2147 DEN1-WP 2100LILEIGKLPQHLTQRAQNALDNLVMLHNSEQGGKAYRHAMEELPDTIETL 2149 DEN2-NGC 2099LITEMGRLPTFMTQKARDALDNLAVLHTAEAGGRAYNHALSELPETLETL 2148 DEN3-H87 2098LVTEIGRVPSHLAHRTRNALDNLVMLHTSEHGGRAYRHAVEELPETMETL 2147.. *.  .*  .. ... ****. .**..* **.** **. ***...***  DEN4 2148MLVALLGAMTAGIFLFFMQGKGIGKLSMGLITIAVASGLLWVAEIQPQWI 2197 DEN1-WP 2150MLLALIAVLTGGVTLFFLSGRGLGKTSIGLLCVIASSALLWMASVEPHWI 2199 DEN2-NGC 2149LLLTLLATVTGGIFLFLMSGRGIGKMTLGMCCIITASILLWYAQIQPHWI 2198 DEN3-H87 2148LLLGLMILLTGGAMLFLISGKGIGKTSIGLICVIASSGMLWMADVPLQWI 2197.*. *.  .* *  ** . *.*.** ..*. ..  .* .** * .  .**  DEN4 2198AASIILEFFLMVLLIPEPEKQRTPQDNQLIYVILTILTIIGLIAANEMGL 2247 DEN1-WP 2200AASIILEFFLMVLLIPEPDRQRTPQDNQLAYVVIGLLFMILTAAANEMGL 2249 DEN2-NGC 2199AASIILEFFLIVLLIPEPEKQRTPQDNQLTYVVIAILTVVAATMANEMGF 2248 DEN3-H87 2198ASAIVLEFFMMVLLIPEPEKQRTPQDNQLAYVVIGILTLAAIVAANEMGL 2247*..*.****..*******..********* **.. .* .     *****  DEN4 2248IEKTKTDFGFY----QVKTETTILDVDLRPASAWTLYAVATTILTPMLRH 2293 DEN1-WP 2250LETTKKDLGIGHAAAENHHHAAMLDVDLHPASAWTLYAVATTIITPMMRH 2299 DEN2-NGC 2249LEKTKKDLGLG-SITTQQPESNILDIDLRPASAWTLYAVATTFVTPMLRH 2297 DEN3-H87 2248LETTKRDLGMS-KEPGVVSPTSYLDVDLHPASAWTLYAVATTVITPMLRH 2296.* ** * *           .  **.**.************* .***.**  DEN4 2294TIENTSANLSLAAIANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVNP 2343 DEN1-WP 2300TIENTTANISLTAIANQAAILMGLDKGWPISKMDIGVPLLALGCYSQVNP 2349 DEN2-NGC 2298SIENSSVNVSLTAIANQATVLMGLGKGWPLSKMDIGVPLLAIGCYSQVNP 2347 DEN3-H87 2297TIENSTANVSLAAIANQAVVLMGLDKGWPISKMDLGVPLLAIGCYSQVNP 2346.***.. *.**.****** .**** ****. .**.******.********  DEN4 2344TTLTASLVMLLVHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGITVID 2393 DEN1-WP 2350LTLTAAVFMLVAHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIVAID 2399 DEN2-NGC 2348ITLTAALFLLVAHYAIIGPGLQAKATREAQKRAAAGIMKNPTVDGITVID 2397 DEN3-H87 2347LTLIAAVLLLVTHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIMTID 2396 ** *.. .*. ********************.*************  **  DEN4 2394LEPISYDPKFEKQLGQVMLLVLCAGQLLLMRTTWAFCEVLTLATGPILTL 2443 DEN1-WP 2400LDPVVYDAKFEKQLGQIMLLILCTSQILLMRTTWALCESITLATGPLTTL 2449 DEN2-NGC 2398LDPIPYDPKFEKQLGQVMLLVLCVTQVLMMRTTWALCEALTLATGPISTL 2447 DEN3-H87 2397LDPVIYDSKFEKQLGQVMLLVLCAVQLLLMRTSWALCEVLTLATGPITTL 2446*.*. ** ********.***.**  *.*.***.** ** .******. **  DEN4 2444WEGNPGRFWNTTIAVSTANIFRGSYLAGAGLAFSLIKNAQTPRRGTGTTG 2493 DEN1-WP 2450WEGSPGKFWNTTIAVSMANIFRGSYLAGAGLAFSLMKSLGGGRRGTGAQG 2499 DEN2-NGC 2448WEGNPGRFWNTTIAVSMANIFRGSYLAGAGLLFSIMKNTTNTRRGTGNIG 2497 DEN3-H87 2447WEGSPGKFWNTTIAVSMANIFRGSYLAGAGLALSIMKSVGTGKRGTGSQG 2496*** **.********* **************  *..*     .****  *  DEN4 2494ETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGSKIKHAVS 2543 DEN1-WP 2500ETLGEKWKRQLNQLSKSEFNTYKRSGIIEVDRSEAKEGLKRGEPTKHAVS 2549 DEN2-NGC 2498ETLGEKWKSRLNALGKSEFQIYKKSGIQEVDRTLAKEGIKRGETDHHAVS 2547 DEN3-H87 2497ETLGEKWKKKLNQLSRKEFDLYKKSGITEVDRTEAKEGLKRGEITHHAVS 2546******** .** * . **  **.*** ****. **  .* *   .****  DEN4 2544RGSSKIRWIVERGMVKPKGKVVDLGCGRGGWSYYMATLKNVTEVKGYTKG 2593 DEN1-WP 2550RGTAKLRWFVERNLVKPEGKVIDLGCGRGGWSYYCAGLKKVTEVKGYTKG 2599 DEN2-NGC 2548RGSAKLRWFVERNMVTPEGKVVDLGCGRGGWSYYCGGLKNVREVKGLTKG 2597 DEN3-H87 2547RGSAKLQWFVERNMVIPEGRVIDLGCGRGGWSYYCAGLKKVTEVRGYTKG 2596**..*..* ***..* * *.*.************   ** * **.* ***  DEN4 2594GPGHEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTI 2643 DEN1-WP 2600GPGHEEPIPMATYGWNLVKLYSGKDVFFTPPEKCDTLLCDIGESSPNPTI 2649 DEN2-NGC 2598GPGHEEPIPMSTYGWNLVRLQSGVDVFFTPPEKCDTLLCDIGESSPNPTV 2647 DEN3-H87 2597GPGHEEPVPMSTYGWNIVKLMSGKDVFYLPPEKCDTLLCDIGESSPSPTV 2646*******.**.*****.*.* ** ***. * *. ***********  **.  DEN4 2644EEGRTLRVLKMVEPWLSSKPEFCIKVLNPYMPTVIEELEKLQRKHGGNLV 2693 DEN1-WP 2650EEGRTLRVLKMVEPWLRGN-QFCIKILNPYMPSVVETLEQMQRKHGGMLV 2698 DEN2-NGC 2648EAGRTLRVLNLVENWLNNNTQFCIKVLNPYMPSVIEKMEALQRKYGGALV 2697 DEN3-H87 2647EESRTIRVLKMVEPWLKNN-QFCIKVLNPYMPTVIEHLERLQRKHGGMLV 2695*  **.*** .** **    .****.******.*.* .* .*** ** **  DEN4 2694RCPLSRNSTHEMYWVSGASGNIVSSVNTTSKMLLNRFTTRHRKPTYEKDV 2743 DEN1-WP 2699RNPLSRNSTHEMYWVSCGTGNIVSAVNMTSRMLLNRFTMAHRKPTYERDV 2748 DEN2-NGC 2698RNPLSRNSTHEMYWVSNASGNIVSSVNMISRMLINRFTMRHKKATYEPDV 2747 DEN3-H87 2696RNPLSRNSTHEMYWISNGTGNIVSSVNMVSRLLLNRFTMTHRRPTIEKDV 2745* ************.*  .*****.**  *..*.****  *.. * * **  DEN4 2744DLGAGTRSVSTETEKPDMTIIGRRLQRLQEEHKETWHYDQENPYRTWAYH 2793 DEN1-WP 2749DLGAGTRHVAVEPEVANLDIIGQRIENIKNGHKSTWHYDEDNPYKTWAYH 2798 DEN2-NGC 2748DLGSGTRNIGIESEIPNLDIIGKRIEKIKQEHETSWHYDQDHPYKTWAYH 2797 DEN3-H87 2746DLGAGTRHVNAEPETPNMDVIGERIKRIKEEHSSTWHYDDENPYKTWAYH 2795***.*** .  * *   . .** *.  ..  *  .**** ..** *****  DEN4 2794GSYEAPSTGSASSMVNGVVKLLTKPWDVIPMVTQLAMTDTTPFGQQRVFK 2843 DEN1-WP 2799GSYEVKPSGSASSMVNGVVRLLTKPWDVIPMVTQIAMTDTTPFGQQRVFK 2848 DEN2-NGC 2798GSYETKQTGSASSMVNGVVRLLTKPWDVVPMVTQMAMTDTTPFGQQRVFK 2847 DEN3-H87 2796GSYEVKATGSASSMINGVVKLLTKPWDVVPMVTQMAMTDTTPFGQQRVFK 2845****   .******.****.********.*****.***************  DEN4 2844EKVDTRTPQPKPGTRMVMTTTANWLWALLGKKKNPRLCTREEFISKVRSN 2893 DEN1-WP 2849EKVDTRTPKAKRGTAQIMEVTARWLWGFLSRNKKPRICTREEFTRKVRSN 2898 DEN2-NGC 2848EKVDTRTQEPKEGTKKLMKITAEWLWKELGKKKTPRMCTREEFTRKVRSN 2897 DEN3-H87 2846EKVDTRTPRPMPGTRKVMEITAEWLWRTLGRNKRPRLCTREEFTKKVRTN 2895*******     **  .*  ** ***  * . * **.******  ***.*  DEN4 2894AAIGAVFQEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMG 2943 DEN1-WP 2899AAIGAVFVDENQWNSAKEAVEDERFWDLVHRERELHKQGKCATCVYNMMG 2948 DEN2-NGC 2898AALGAIFTDENKWKSAREAVEDSRFWELVDKERNLHLEGKCETCVYNMMG 2947 DEN3-H87 2896AAMGAVFTEENQWDSARAAVEDEEFWKLVDRERELHKLGKCGSCVYNMMG 2945**.**.* .*. * **  ** *  ** ** .** **  *** .*******  DEN4 2944KREKKLGEFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSWSG 2993 DEN1-WP 2949KREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFMNEDHWFSRENSLSG 2998 DEN2-NGC 2948KREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFLNEDHWFSRENSLSG 2997 DEN3-H87 2946KREKKLGEFGKAKGSRAIWYMWLGARYLEFEALGFLNEDHWFSRENSYSG 2995**********.***************.********.****** **** **  DEN4 2994VEGEGLHRLGYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQ 3043 DEN1-WP 2999VEGEGLHKLGYILRDISKIPGGNMYADDTAGWDTRITEDDLQNEAKITDI 3048 DEN2-NGC 2998VEGEGLHKLGYILRDVSKKEGGAMYADDTAGWDTRITLEDLKNEEMVTNH 3047 DEN3-H87 2996VEGEGLHKLGYILRDISKIPGGAMYADDTAGWDTRITEDDLHNEEKITQQ 3045*******.***** .. *  *  ************** .**.**  .*  DEN4 3044MAPHHKILAKAIFKLTYQNKVVKVLRPTPRGAVMDIISRKDQRGSGQVGT 3093 DEN1-WP 3049MEPEHALLATSIFKLTYQNKVVRVQRPAKNGTVMDVISRRDQRGSGQVGT 3098 DEN2-NGC 3048MEGEHKKLAEAIFKLTYQNKVVRVQRPTPRGTVMDIISRRDQRGSGQVGT 3097 DEN3-H87 3046MDPEHRQLANAIFKLTYQNKVVKVQRPTPKGTVMDIISRKDQRGSGQVGT 3095*   *  ** .***********.* **.  *.***.***.**********  DEN4 3094YGLNTFTNMEVQLIRQMEAEGVITQDDMQNPKGLKERVEKWLKECGVDRL 3143 DEN1-WP 3099YGLNTFTNMEAQLIRQMESEGIFSPSELETPN-LAERVLDWLKKHGTERL 3147 DEN2-NGC 3098YGLNTFTNMEAQLIRQMEGEGVFKSIQHLTVT-EEIAVQNWLARVGRERL 3146 DENS-H87 3096YGLNTFTNMEAQLIRQMEGEGVLSKADLENPHPLEKKITQWLETKGVERL 3145********** ******* **.       .       .  **   * .**  DEN4 3144KRMAISGDDCVVKPLDERFGTSLLFLNDMGKVRKDIPQWEPSKGWKNWQE 3193 DEN1-WP 3148KRMAISGDDCVVKPIDDRFATALTALNDMGKVRKDIPQWEPSKGWNDWQQ 3197 DEN2-NGC 3147SRMAISGDDCVVKPLDDRFASALTALNDMGKVRKDIQQWEPSRGWNDWTQ 3196 DENS-H87 3146KRMAISGDDCVVKPIDDRFANALLALNDMGKVRKDIPQWQPSKGWHDWQQ 3195 *************.*.**  .*  *********** **.**.**  * . DEN4 3194VPFCSHHFHKIFMKDGRSLVVPCRNQDELIGRARISQGAGWSLRETACLG 3243 DEN1-WP 3198VPFCSHHFHQLIMKDGREIVVPCRNQDELVGRARVSQGAGWSLRETACLG 3247 DEN2-NGC 3197VPFCSHHFHELIMKDGRVLVVPCRNQDELIGRARISQGAGWSLRETACLG 3246 DENS-H87 3196VPFCSHHFHELIMKDGRKLVVPCRPQDELIGRARISQGAGWSLRETACLG 3245********* . ***** .***** ****.****.*************** DEN4 3244KAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWSIHAHHQWMT 3293 DEN1-WP 3248KSYAQMWQLMYFHRRDLRLAANAICSAVPVDWVPTSRTTWSIHAHHQWMT 3297 DEN2-NGC 3247KSYAQMWSLMYFHRRDLRLAANAICSAVPSHWVPTSRTTWSIHAKHEWMT 3296 DENS-H87 3246KAYAQMWTLMYFHRRDLRLASNAICSAVPVHWVPTSRTTWSIHAHHQWMT 3295*.***** ************. *******  * ***********.*.*** DEN4 3294TEDMLKVWNRVWIEDNPNMTDKTPVHSWEDIPYLGKREDLWCGSLIGLSS 3343 DEN1-WP 3298TEDMLSVWNRVWIEENPWMEDKTHVSSWEDVPYLGKREDRWCGSLIGLTA 3347 DEN2-NGC 3297TEDMLTVWNRVWIQENPWMEDKTPVESWEEIPYLGKREDQWCGSLIGLTS 3346 DENS-H87 3296TEDMLTVWNRVWIEDNPWMEDKTPVTTWEDVPYLGKREDQWCGSLIGLTS 3345***** *******..** * *** * .**..******** ********.. DEN4 3344RATWAKNIHTAITQVRNLIGKEEYVDYMPVMKRYSAPSESEGVL 3387 DEN1-WP 3348RATWATNIQVAINQVRRLIGNENYLDFMTSMKRFKNESDPEGALW 3392 DEN2-NGC 3347RATWAKNIQTAINQVRSLIGNEEYTDYMPSMKRFRREEEEAGVLW 3391 DENS-H87 3346RATWAQNILTAIQQVRSLIGNEEFLDYMPSMKRFRKEEESEGAIW 3390***** **  ** *** *** * . *.* ***.     .  * .  *Residue identity .Residuesimilarity

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

What is claimed is:
 1. An attenuated mutant dengue (DEN) viruscomprising: a proline to leucine change at amino acid position 2343 ofnon-structural protein 4B (NS4B), wherein the numbering is based uponthe prototypic isolate DEN4 Dominica 1981 and the attenuated mutant DENvirus has at least one of the following properties: improved replicationin Vero cells or restricted replication in mosquito cells.
 2. Theattenuated mutant DEN virus of claim 1, further comprising a 30nucleotide deletion in the 3′ untranslated region (UTR) of the viralgenome (designated Δ30), wherein the 30 nucleotide deletion correspondsto nucleotides 172-143 of the prototypic isolate DEN4 Dominica
 1981. 3.The attenuated mutant DEN virus of claim 1, wherein the attenuatedmutant DEN virus is a dengue virus selected from the group consisting ofdengue virus type 1, dengue virus type 2, dengue virus type 3, anddengue virus type
 4. 4. The attenuated mutant DEN virus of claim 1,wherein the attenuated mutant DEN virus is a chimeric virus.
 5. Thechimeric virus of claim 4 having a backbone selected from the groupconsisting of a dengue 1 backbone, a dengue 2 backbone, a dengue 3backbone, and a dengue 4 backbone.
 6. The attenuated mutant DEN virus ofclaim 1, wherein the attenuated mutant DEN virus phenotype is nottemperature sensitivity in Vero cells or the human liver cell lineHuH-7.
 7. The attenuated mutant DEN virus of claim 1, wherein theattenuated mutant DEN virus is does not display attenuation in mice. 8.A pharmaceutical composition comprising a pharmacologically acceptablevehicle and the attenuated mutant DEN virus according to claim
 1. 9. Akit comprising a pharmaceutical composition according to claim 8 in apack or dispenser device and instructions for administration.
 10. Amethod of producing neutralizing antibodies against dengue viruscomprising the administration of a therapeutically effective amount of apharmaceutical composition comprising a pharmacologically acceptablevehicle and the attenuated mutant DEN virus according to claim
 1. 11.The method of claim 10, wherein administration is by subcutaneous,intradermal, or intramuscular injection.
 12. An immunogenic compositioncomprising: a pharmacologically acceptable vehicle and an attenuatedmutant dengue (DEN) virus comprising a proline to leucine change atamino acid position 2343 of non-structural protein 4B (NS4B), whereinthe numbering is based upon the prototypic isolate DEN4 Dominica 1981and the attenuated mutant DEN virus has at least one of the followingproperties: improved replication in Vero cells or restricted replicationin mosquito cells.
 13. The immunogenic composition of claim 12 in unitdosage form having from about 10²-10⁹ plaque forming units (PFU)/ml. 14.An immunogenic composition comprising a pharmacologically acceptablevehicle and the attenuated mutant DEN virus according to claim
 1. 15.The immunogenic composition of claim 14 in unit dosage form having fromabout 0.1 to 50 μg of E protein/ml.
 16. A method of preparing anattenuated mutant DEN virus comprising: (a) synthesizing full-lengthviral genomic RNA in vitro using a cDNA molecule that encodes anattenuated mutant DEN virus according to claim 1; (b) transfectingcultured cells with the viral genomic RNA to produce virus; and (c)isolating the virus from the cultured cells.
 17. A method of making apharmaceutical composition comprising combining a pharmacologicallyacceptable vehicle and the attenuated mutant DEN virus according toclaim 1.